Stents having bioabsorbable layers

ABSTRACT

Provided herein is a device comprising: a. stent; b. a plurality of layers on said stent framework to form said device; wherein at least one of said layers comprises a bioabsorbable polymer and at least one of said layers comprises one or more active agents; wherein at least part of the active agent is in crystalline form.

CROSS REFERENCE

This application claims the benefit of U.S. Provisional Application No.61/045,928, filed Apr. 17, 2008, and U.S. Provisional Application No.61/104,669 filed Oct. 10, 2008. The contents of the applications areincorporated herein by reference in their entirety.

This application relates to U.S. Provisional Application No. 60/912,408,filed Apr. 17, 2007, U.S. Provisional Application No. 60/912,394, filedApr. 17, 2007, U.S. Provisional Application No. 60/981,445, filed Oct.19, 2007 and U.S. Provisional Application entitled Stents HavingBioabsorbable Layers, Attorney Docket No. 32605-704-108, filed Apr. 17,2009. The contents of the applications are incorporated herein byreference in their entirety.

BACKGROUND OF THE INVENTION

Drug-eluting stents are used to address the drawbacks of bare stents,namely to treat restenosis and to promote healing of the vessel afteropening the blockage by PCI/stenting. Some current drug eluting stentscan have physical, chemical and therapeutic legacy in the vessel overtime. Others may have less legacy, bur are not optimized for thickness,deployment flexibility, access to difficult lesions, and minimization ofvessel wall intrusion.

SUMMARY OF THE INVENTION

The present invention relates to methods for forming stents comprising abioabsorbable polymer and a pharmaceutical or biological agent in powderform onto a substrate.

It is desirable to have a drug-eluting stent with minimal physical,chemical and therapeutic legacy in the vessel after a proscribed periodof time. This period of time is based on the effective healing of thevessel after opening the blockage by PCI/stenting (currently believed byleading clinicians to be 6-18 months).

It is also desirable to have drug-eluting stents of minimalcross-sectional thickness for (a) flexibility of deployment (b) accessto small vessels (c) minimized intrusion into the vessel wall and blood.

Provided herein is a device comprising a stent; and a plurality oflayers that form a laminate coating on said stent; wherein at least oneof said layers comprises a bioabsorbable polymer and at least one ofsaid layers comprises one or more active agents; wherein at least aportion of the active agent is in crystalline form.

Provided herein is a device comprising a stent; and a plurality oflayers that form a laminate coating on said stent; wherein at least oneof said layers comprises a bioabsorbable polymer and at least one ofsaid layers comprises a pharmaceutical agent selected from rapamycin, aprodrug, a derivative, an analog, a hydrate, an ester, and a saltthereof, wherein at least a portion of the pharmaceutical agent is incrystalline form.

In some embodiments, the device has at least one pharmaceutical agentlayer defined by a three-dimensional physical space occupied by crystalparticles of said pharmaceutical agent and said three dimensionalphysical space is free of polymer. In some embodiments, at least some ofthe crystal particles in said three dimensional physical space definingsaid at least one pharmaceutical agent layer are in contact with polymerparticles present in a polymer layer adjacent to said at least onepharmaceutical agent layer defined by said three-dimensional space freeof polymer.

In some embodiments, the plurality of layers comprises a first polymerlayer comprising a first bioabsorbable polymer and a second polymerlayer comprising a second bioabsorbable polymer, wherein said at leastone layer comprising said pharmaceutical agent is between said firstpolymer layer and said second polymer layer. In some embodiments, firstand second bioabsorbable polymers are the same polymer. In someembodiments, the first and second bioabsorbable polymers are different.In some embodiments, the second polymer layer has at least one contactpoint with at least one particle of said pharmaceutical agent in saidpharmaceutical agent layer and said second polymer layer has at leastone contact point with said first polymer layer.

In some embodiments, the stent has a stent longitudinal axis; and saidsecond polymer layer has a second polymer layer portion along said stentlongitudinal wherein said second layer portion is free of contact withparticles of said pharmaceutical agent. In some embodiments, the devicehas at least one pharmaceutical agent layer defined by athree-dimensional physical space occupied by crystal particles of saidpharmaceutical agent and said three dimensional physical space is freeof polymer.

In some embodiments, the stent comprises at least one strut having astrut length along said stent longitudinal axis, wherein said secondlayer portion extends substantially along said strut length. In someembodiments, the stent has a stent length along said stent longitudinalaxis and said second layer portion extends substantially along saidstent length.

In some embodiments, the stent comprises at least five struts, eachstrut having a strut length along said stent longitudinal axis, whereinsaid second layer portion extends substantially along substantially thestrut length of at least two struts. In some embodiments, the stentcomprises at least five struts, each strut having a strut length alongsaid stent longitudinal axis, wherein said second layer portion extendssubstantially along substantially the strut length of at least threestruts. In some embodiments, the stent comprises at least five struts,each strut having a strut length along said stent longitudinal axis,wherein said second layer portion extends substantially alongsubstantially the strut length of least four struts. In someembodiments, the stent comprises at least five struts, each strut havinga strut length along said stent longitudinal axis, wherein said secondlayer portion extends substantially along substantially the strut lengthof all said at least five struts. In some embodiments, the stent has astent length along said stent longitudinal axis and said second layerportion extends substantially along said stent length.

In some embodiments, the stent has a stent length along said stentlongitudinal axis and said second layer portion extends along at least50% of said stent length. In some embodiments, the stent has a stentlength along said stent longitudinal axis and said second layer portionextends along at least 75% of said stent length. In some embodiments,the stent has a stent length along said stent longitudinal axis and saidsecond layer portion extends along at least 85% of said stent length. Insome embodiments, the stent has a stent length along said stentlongitudinal axis and said second layer portion extends along at least90% of said stent length. In some embodiments, the stent has a stentlength along said stent longitudinal axis and said second layer portionextends along at least 99% of said stent length.

In some embodiments, the laminate coating has a total thickness and saidsecond polymer layer portion has a thickness of from about 0.01% toabout 10% of the total thickness of said laminate coating. In someembodiments, the laminate coating has a total thickness and saidhorizontal second polymer layer portion has a thickness of from about 1%to about 5% of the total thickness of said laminate coating. In someembodiments, the laminate coating has a total thickness of from about 5μm to about 50 μm and said horizontal second polymer layer portion has athickness of from about 0.001 μm to about 5 μm. In some embodiments, thelaminate coating has a total thickness of from about 10 μm to about 20μm and said second polymer layer portion has a thickness of from about0.01 μm to about 5 μm.

In some embodiments, the laminate coating is at least 25% by volumepharmaceutical agent. In some embodiments, the laminate coating is atleast 35% by volume pharmaceutical agent. In some embodiments, thelaminate coating is about 50% by volume pharmaceutical agent.

In some embodiments, at least a portion of the pharmaceutical agent ispresent in a phase separate from one or more phases formed by saidpolymer.

In some embodiments, the pharmaceutical agent is at least 50%crystalline. In some embodiments, the pharmaceutical agent is at least75% crystalline. In some embodiments, the pharmaceutical agent is atleast 90% crystalline. In some embodiments, the pharmaceutical agent isat least 95% crystalline. In some embodiments, the pharmaceutical agentis at least 99% crystalline.

In some embodiments, the stent has a stent longitudinal length and thecoating has a coating outer surface along said stent longitudinallength, wherein said coating comprises pharmaceutical agent incrystalline form present in the coating below said coating outersurface. In some embodiments, the stent has a stent longitudinal lengthand the coating has a coating outer surface along said stentlongitudinal length, wherein said coating comprises pharmaceutical agentin crystalline form present in the coating up to at least 1 μm belowsaid coating outer surface. In some embodiments, the stent has a stentlongitudinal length and the coating has a coating outer surface alongsaid stent longitudinal length, wherein said coating comprisespharmaceutical agent in crystalline form present in the coating up to atleast 5 μm below said coating outer surface.

In some embodiments, the coating exhibits an X-ray spectrum showing thepresence of said pharmaceutical agent in crystalline form. In someembodiments, the coating exhibits a Raman spectrum showing the presenceof said pharmaceutical agent in crystalline form. In some embodiments,the coating exhibits a Differential Scanning Calorimetry (DSC) curveshowing the presence of said pharmaceutical agent in crystalline form.The device of claims 36-38, wherein said coating exhibits Wide AngleX-ray Scattering (WAXS) spectrum showing the presence of saidpharmaceutical agent in crystalline form. In some embodiments, thecoating exhibits a wide angle radiation scattering spectrum showing thepresence of said pharmaceutical agent in crystalline form. In someembodiments, the coating exhibits an Infra Red (IR) spectrum showing thepresence of said pharmaceutical agent in crystalline form.

In some embodiments, the stent has a stent longitudinal axis and a stentlength along said stent longitudinal axis, wherein said coating isconformal to the stent along substantially said stent length.

In some embodiments, the stent has a stent longitudinal axis and a stentlength along said stent longitudinal axis, wherein said coating isconformal to the stent along at least 75% of said stent length. In someembodiments, the stent has a stent longitudinal axis and a stent lengthalong said stent longitudinal axis, wherein said coating is conformal tothe stent along at least 85% of said stent length. In some embodiments,the stent has a stent longitudinal axis and a stent length along saidstent longitudinal axis, wherein said coating is conformal to the stentalong at least 90% of said stent length. In some embodiments, the stenthas a stent longitudinal axis and a stent length along said stentlongitudinal axis, wherein said coating is conformal to the stent alongat least 95% of said stent length. In some embodiments, the stent has astent longitudinal axis and a stent length along said stent longitudinalaxis, wherein said coating is conformal to the stent along at least 99%of said stent length.

In some embodiments, the stent has a stent longitudinal axis and aplurality of struts along said stent longitudinal axis, wherein saidcoating is conformal to at least 50% of said struts. In someembodiments, the stent has a stent longitudinal axis and a plurality ofstruts along said stent longitudinal axis, wherein said coating isconformal to at least 75% of said struts. In some embodiments, the stenthas a stent longitudinal axis and a plurality of struts along said stentlongitudinal axis, wherein said coating is conformal to at least 90% ofsaid struts. In some embodiments, the stent has a stent longitudinalaxis and a plurality of struts along said stent longitudinal axis,wherein said coating is conformal to at least 99% of said struts. Insome embodiments, the stent has a stent longitudinal axis and a stentlength along said stent longitudinal axis, wherein an electronmicroscopy examination of the device shows said coating is conformal tosaid stent along at least 90% of said stent length.

In some embodiments, the stent has a stent longitudinal axis and a stentlength along said stent longitudinal axis, wherein said coating has asubstantially uniform thickness along substantially said stent length.

In some embodiments, the stent has a stent longitudinal axis and a stentlength along said stent longitudinal axis, wherein said coating has asubstantially uniform thickness along at least 75% of said stent length.In some embodiments, the stent has a stent longitudinal axis and a stentlength along said stent longitudinal axis, wherein said coating has asubstantially uniform thickness along at least 95% of said stent length.

In some embodiments, the stent has a stent longitudinal axis and a stentlength along said stent longitudinal axis, wherein said coating has anaverage thickness determined by an average calculated from coatingthickness values measured at a plurality of points along said stentlongitudinal axis; wherein a thickness of the coating measured at anypoint along stent longitudinal axis is from about 75% to about 125% ofsaid average thickness. In some embodiments, the stent has a stentlongitudinal axis and a stent length along said stent longitudinal axis,wherein said coating has an average thickness determined by an averagecalculated from coating thickness values measured at a plurality ofpoints along said stent longitudinal axis; wherein a thickness of thecoating measured at any point along stent longitudinal axis is fromabout 95% to about 105% of said average thickness.

Provided herein is a device comprising: a stent; and a plurality oflayers that form a laminate coating on said stent, wherein a first layercomprises a first bioabsorbable polymer, a second layer comprises apharmaceutical agent, a third layer comprises a second bioabsorbablepolymer, a fourth layer comprises the pharmaceutical agent, and a fifthlayer comprises a third bioabsorbable polymer, wherein thepharmaceutical agent is selected from rapamycin, a prodrug, aderivative, an analog, a hydrate, an ester, and a salt thereof, andwherein at least a portion of the pharmaceutical agent is in crystallineform.

In some embodiments, at least two of said first bioabsorbable polymer,said second bioabsorbable polymer and said third bioabsorbable polymerare the same polymer. In some embodiments, the first bioabsorbablepolymer, the second bioabsorbable polymer and the third bioabsorbablepolymer are the same polymer. In some embodiments, at least two of saidfirst bioabsorbable polymer, said second bioabsorbable polymer and saidthird bioabsorbable polymer are different polymers. In some embodiments,the first bioabsorbable polymer, said second bioabsorbable polymer andsaid third bioabsorbable polymer are different polymers.

In some embodiments, the third layer has at least one contact point withparticles of said pharmaceutical agent in said second layer; and saidthird layer has at least one contact point with said first layer.

In some embodiments, at least two of the first polymer, the secondpolymer, and the third polymer are the same polymer, and wherein saidsame polymer comprises a PLGA copolymer. In some embodiments, the thirdpolymer has an in vitro dissolution rate higher than the in vitrodissolution rate of the first polymer. In some embodiments, the thirdpolymer is PLGA copolymer with a ratio of about 40:60 to about 60:40 andthe first polymer is a PLGA copolymer with a ratio of about 70:30 toabout 90:10. In some embodiments, the third polymer is PLGA copolymerhaving a molecular weight of about 10 kD and the second polymer is aPLGA copolymer having a molecular weight of about 19 kD.

In some embodiments, measuring the in vitro dissolution rate of saidpolymers comprises contacting the device with elution media anddetermining polymer weight loss at one or more selected time points. Insome embodiments, measuring the in vitro dissolution rate of saidpolymers comprises contacting the device with elution media anddetermining polymer weight loss at one or more selected time points.

Provided herein is a device, comprising: a stent; and a coating on saidstent comprising a first bioabsorbable polymer, a second bioabsorbablepolymer; and pharmaceutical agent selected from rapamycin, a prodrug, aderivative, an analog, a hydrate, an ester, and a salt thereof whereinat least a portion of the pharmaceutical agent is in crystalline form,and wherein the first polymer has an in vitro dissolution rate higherthan the in vitro dissolution rate of the second polymer.

In some embodiments, the first polymer is PLGA copolymer with a ratio ofabout 40:60 to about 60:40 and the second polymer is a PLGA copolymerwith a ratio of about 70:30 to about 90:10. In some embodiments, thefirst polymer is PLGA copolymer having a molecular weight of about 10 kDand the second polymer is a PLGA copolymer having a molecular weight ofabout 19 kD. In some embodiments, measuring the in vitro dissolutionrate of said polymers comprises contacting the device with elution mediaand determining polymer weight loss at one or more selected time points.

Provided herein is a device comprising a stent; and a plurality oflayers that form a laminate coating on said stent; wherein at least oneof said layers comprises a first bioabsorbable polymer, at least one ofsaid layers comprises a second bioabsorbable polymer, and at least oneof said layers comprises one or more active agents; wherein at least aportion of the active agent is in crystalline form, and wherein thefirst polymer has an in vitro dissolution rate higher than the in vitrodissolution rate of the second polymer.

Provided herein is a device comprising a stent; and a plurality oflayers that form a laminate coating on said stent; wherein at least oneof said layers comprises a first bioabsorbable polymer, at least one ofsaid layers comprises a second bioabsorbable polymer, and at least oneof said layers comprises a pharmaceutical agent selected from rapamycin,a prodrug, a derivative, an analog, a hydrate, an ester, and a saltthereof, wherein at least a portion of the pharmaceutical agent is incrystalline form and wherein the first polymer has an in vitrodissolution rate higher than the in vitro dissolution rate of the secondpolymer.

In some embodiments, the first polymer is PLGA copolymer with a ratio ofabout 40:60 to about 60:40 and the second polymer is a PLGA copolymerwith a ratio of about 70:30 to about 90:10. In some embodiments, thefirst polymer is PLGA copolymer having a molecular weight of about 10 kDand the second polymer is a PLGA copolymer having a molecular weight ofabout 19 kD. In some embodiments, measuring the in vitro dissolutionrate comprises contacting the device with elution media and determiningpolymer weight loss at one or more selected time points.

Provided herein is a device comprising a stent; and a plurality oflayers that form a laminate coating on said stent; wherein at least oneof said layers comprises a bioabsorbable polymer, at least one of saidlayers comprises a first active agent and at least one of said layerscomprises a second active agent; wherein at least a portion of firstand/or second active agents is in crystalline form.

In some embodiments, the bioabsorbable polymer is selected from thegroup PLGA, PGA poly(glycolide), LPLA poly(1-lactide), DLPLApoly(dl-lactide), PCL poly(∈-caprolactone) PDO, poly(dioxolane) PGA-TMC,85/15 DLPLG p(dl-lactide-co-glycolide), 75/25 DLPL, 65/35 DLPLG, 50/50DLPLG, TMC poly(trimethylcarbonate), p(CPP:SA)poly(1,3-bis-p-(carboxyphenoxy)propane-co-sebacic acid). In someembodiments, the polymer comprises an intimate mixture of two or morepolymers.

In some embodiments, the first and second active agents areindependently selected from pharmaceutical agents and active biologicalagents.

In some embodiments, the stent is formed of stainless steel material. Insome embodiments, the stent is formed of a material comprising a cobaltchromium alloy. In some embodiments, the stent is formed from a materialcomprising the following percentages by weight: about 0.05 to about 0.15C, about 1.00 to about 2.00 Mn, about 0.04 Si, about 0.03 P, about 0.3S, about 19.0 to about 21.0 Cr, about 9.0 to about 11.0 Ni, about 14.0to about 16.00 W, about 3.0 Fe, and Bal. Co. In some embodiments, thestent is formed from a material comprising at most the followingpercentages by weight: about 0.025 C, about 0.15 Mn, about 0.15 Si,about 0.015 P, about 0.01 S, about 19.0 to about 21.0 Cr, about 33 toabout 37 Ni, about 9.0 to about 10.5 Mo, about 1.0 Fe, about 1.0 Ti, andBal. Co. In some embodiments, the stent is formed from a materialcomprising L605 alloy.

In some embodiments, the stent has a thickness of from about 50% toabout 90% of a total thickness of said device. In some embodiments, thedevice has a thickness of from about 20 μm to about 500 μm. In someembodiments, the device has a thickness of about 90 μm or less. In someembodiments, the laminate coating has a thickness of from about 5 μm toabout 50 μm. In some embodiments, the laminate coating has a thicknessof from about 10 μm to about 20 μm. In some embodiments, the stent has athickness of from about 50 μm to about 80 μm.

Provided herein is a device comprising: a stent, wherein the stent isformed from a material comprising the following percentages by weight:0.05-0.15 C, 1.00-2.00 Mn, 0.040 Si, 0.030 P, 0.3 S, 19.00-21.00 Cr,9.00-11.00 Ni, 14.00-16.00 W, 3.00 Fe, and Bal. Co; and a plurality oflayers that form a laminate coating on said stent, wherein a first layercomprises a first bioabsorbable polymer, a second layer comprises apharmaceutical agent, a third layer comprises a second bioabsorbablepolymer, a fourth layer comprises the pharmaceutical agent, and a fifthlayer comprises a third bioabsorbable polymer, wherein thepharmaceutical agent is selected from rapamycin, a prodrug, aderivative, an analog, a hydrate, an ester, and a salt thereof, whereinat least a portion of the pharmaceutical agent is in crystalline form,and wherein at least one of said first polymer, second polymer and thirdpolymer comprises a PLGA copolymer.

In some embodiments, the device has a pharmaceutical agent content offrom about 0.5 μg/mm to about 20 μg/mm. In some embodiments, the devicehas a pharmaceutical agent content of from about 8 μg/mm to about 12μg/mm. In some embodiments, the device has a pharmaceutical agentcontent of from about 5 μg to about 500 μg. In some embodiments, thedevice has a pharmaceutical agent content of from about 100 μg to about160 μg. In some embodiments, the device has a pharmaceutical agentcontent of from about 100 μg to about 160 μg.

Provided herein is a method of preparing a device comprising a stent anda plurality of layers that form a laminate coating on said stent; saidmethod comprising: (a) providing a stent; (b) forming a plurality oflayers on said stent to form said laminate coating on said stent;wherein at least one of said layers comprises a bioabsorbable polymerand at least one of said layers comprises one or more active agents;wherein at least a portion of the active agent is in crystalline form.

Provided herein is a method of preparing a device comprising a stent anda plurality of layers that form a laminate coating on said stent; saidmethod comprising: (a) providing a stent; (b) forming a plurality oflayers to form said laminate coating on said stent; wherein at least oneof said layers comprises a bioabsorbable polymer and at least one ofsaid layers comprises a pharmaceutical agent selected from rapamycin, aprodrug, a derivative, an analog, a hydrate, an ester, and a saltthereof, wherein at least a portion of the pharmaceutical agent is incrystalline form.

Provided herein is a method of preparing a device comprising a stent anda plurality of layers that form a laminate coating on said stent; saidmethod comprising: (a) providing a stent; (b) forming a plurality oflayers to form said laminate coating on said stent; wherein at least oneof said layers comprises a bioabsorbable polymer and at least one ofsaid layers comprises a pharmaceutical agent selected from rapamycin, aprodrug, a derivative, an analog, a hydrate, an ester, and a saltthereof, wherein at least a portion of the pharmaceutical agent is incrystalline form, wherein said method comprises forming at least onepharmaceutical agent layer defined by a three-dimensional physical spaceoccupied by crystal particles of said pharmaceutical agent and saidthree dimensional physical space is free of polymer.

Provided herein is a method of preparing a device comprising a stent anda plurality of layers that form a laminate coating on said stent; saidmethod comprising: (a) providing a stent; (b) discharging at least onepharmaceutical agent and/or at least one active biological agent in drypowder form through a first orifice; (c) forming a supercritical or nearsupercritical fluid solution comprising at least one supercritical fluidsolvent and at least one polymer and discharging said supercritical ornear supercritical fluid solution through a second orifice underconditions sufficient to form solid particles of the polymer; (d)depositing the polymer and pharmaceutical agent and/or active biologicalagent particles onto said substrate, wherein an electrical potential ismaintained between the substrate and the polymer and pharmaceuticalagent and/or active biological agent particles, thereby forming saidcoating; and (e) sintering said polymer under conditions that do notsubstantially modify a morphology of said pharmaceutical agent and/oractivity of said biological agent.

In some embodiments, step (b) comprises discharging a pharmaceuticalagent selected from rapamycin, a prodrug, a derivative, an analog, ahydrate, an ester, and a salt thereof, wherein at least a portion of thepharmaceutical agent is in crystalline form. In some embodiments, step(c) comprises forming solid particles of a bioabsorbable polymer.

In some embodiments, step (e) comprises forming a polymer layer having alength along a horizontal axis of said device wherein said polymer layerhas a layer portion along said length, wherein said layer portion isfree of pharmaceutical agent.

In some embodiments, step (e) comprises contacting said polymer with adensified fluid. In some embodiments, step (e) comprises contacting saidpolymer with a densified fluid for a period of time at a temperature offrom about 5° C. and 150° C. and a pressure of from about 10 psi toabout 500 psi. In some embodiments, step (e) comprises contacting saidpolymer with a densified fluid for a period of time at a temperature offrom about 25° C. and 95° C. and a pressure of from about 25 psi toabout 100 psi. In some embodiments, step (e) comprises contacting saidpolymer with a densified fluid for a period of time at a temperature offrom about 50° C. and 85° C. and a pressure of from about 35 psi toabout 65 psi.

Provided herein is a method of preparing a device comprising a stent anda plurality of layers that form a laminate coating on said stent; saidmethod comprising: (a) providing a stent; (b) forming a supercritical ornear supercritical fluid solution comprising at least one supercriticalfluid solvent and a first polymer, discharging said supercritical ornear supercritical fluid solution under conditions sufficient to formsolid particles of said first polymer, depositing said first polymerparticles onto said stent, wherein an electrical potential is maintainedbetween the stent and the first polymer, and sintering said firstpolymer; (c) depositing pharmaceutical agent particles in dry powderform onto said stent, wherein an electrical potential is maintainedbetween the stent and said pharmaceutical agent particles; and (d)forming a supercritical or near supercritical fluid solution comprisingat least one supercritical fluid solvent and a second polymer anddischarging said supercritical or near supercritical fluid solutionunder conditions sufficient to form solid particles of said secondpolymer, wherein an electrical potential is maintained between the stentand the second polymer, and sintering said second polymer.

In some embodiments, step (c) and step (d) are repeated at least once.In some embodiments, steps (c) and step (d) are repeated 2 to 20 times.

In some embodiments, the pharmaceutical agent is selected fromrapamycin, a prodrug, a derivative, an analog, a hydrate, an ester, anda salt thereof, wherein at least a portion of the pharmaceutical agentis in crystalline form. In some embodiments, the first and secondpolymers are bioabsorbable.

In some embodiments, step (d) comprises forming a polymer layer having alength along a horizontal axis of said device wherein said polymer layerhas a layer portion along said length, wherein said layer portion isfree of pharmaceutical agent.

In some embodiments, sintering said first and/or sintering said secondpolymer comprises contacting said first and/or second polymer with adensified fluid.

In some embodiments, the contacting step is carried out for a period offrom about 1 minute to about 60 minutes. In some embodiments, thecontacting step is carried out for a period of from about 10 minutes toabout 30 minutes.

In some embodiments, maintaining said electrical potential between saidpolymer particles and or pharmaceutical agent particles and said stentcomprises maintaining a voltage of from about 5 kvolts to about 100kvolts. In some embodiments, maintaining said electrical potentialbetween said polymer particles and or pharmaceutical agent particles andsaid stent comprises maintaining a voltage of from about 20 kvolts toabout 30 kvolts.

Provided herein is a device prepared by a process comprising a method asdescribed herein.

Provided herein is method of treating a subject comprising delivering adevice as described herein in a body lumen of the subject.

Provided herein is a method of treating a subject comprising deliveringin the body of the subject a device comprising: a stent, wherein thestent is formed from a material comprising the following percentages byweight: 0.05-0.15 C, 1.00-2.00 Mn, 0.040 Si, 0.030 P, 0.3 S, 19.00-21.00Cr, 9.00-11.00 Ni, 14.00-16.00 W, 3.00 Fe, and Bal. Co; and a pluralityof layers that form a laminate coating on said stent, wherein a firstlayer comprises a first bioabsorbable polymer, a second layer comprisesa pharmaceutical agent, a third layer comprises a second bioabsorbablepolymer, a fourth layer comprises the pharmaceutical agent, and a fifthlayer comprises a third bioabsorbable polymer, wherein thepharmaceutical agent is selected from rapamycin, a prodrug, aderivative, an analog, a hydrate, an ester, and a salt thereof, whereinat least a portion of the pharmaceutical agent is in crystalline form,and wherein at least one of said first polymer, second polymer and thirdpolymer comprises a PLGA copolymer.

In some embodiments, the device has a pharmaceutical agent content offrom about 0.5 μg/mm to about 20 μg/mm. In some embodiments, the devicehas a pharmaceutical agent content of from about 8 μg/mm to about 12μg/mm. In some embodiments, the device has a pharmaceutical agentcontent of from about 100 μg to about 160 μg. In some embodiments, thedevice has a pharmaceutical agent content of from about 120 μg to about150 μg.

In some embodiments, the device has an initial pharmaceutical agentamount and the amount of pharmaceutical agent delivered by said deviceto vessel wall tissue of said subject is higher than the amount ofpharmaceutical agent delivered by a conventional drug eluting stenthaving the same initial pharmaceutical agent content as the initialpharmaceutical agent content of said device. In some embodiments, theamount of pharmaceutical agent delivered by said device to vessel walltissue of said subject is at least 25% more that the amount ofpharmaceutical agent delivered to vessel wall tissue of said subject bysaid conventional drug eluting stent. In some embodiments, the methodcomprises treating restenosis in a blood vessel of said the subject. Insome embodiments, the subject is selected from a pig, a rabbit and ahuman.

Provided herein is a device comprising: a stent; and a plurality oflayers on said stent; wherein at least one of said layers comprises abioabsorbable polymer and at least one of said layers comprises apharmaceutical agent selected from rapamycin, a prodrug, a derivative,an analog, a hydrate, an ester, and a salt thereof, wherein said deviceprovides an in vitro pharmaceutical agent elution profile wherein saidelution profile shows about 5% to about 25% of pharmaceutical agent iseluted one day after the device is contacted with elution media; 15% toabout 45% of pharmaceutical agent is eluted 7 days after the device iscontacted with elution media; about 25% to about 60% of pharmaceuticalagent is eluted 14 days after the device is contacted with elutionmedia; about 35% to about 70% of pharmaceutical agent is eluted 21 daysafter the device is contacted with elution media; and about 40% to about100% of pharmaceutical agent is eluted 28 days after the device iscontacted with elution media.

Provided herein is a device comprising a stent; and a plurality oflayers on said stent; wherein at least one of said layers comprises abioabsorbable polymer and at least one of said layers comprises apharmaceutical agent selected from rapamycin, a prodrug, a derivative,an analog, a hydrate, an ester, and a salt thereof, wherein said deviceprovides an in vitro pharmaceutical agent elution profile wherein saidelution profile shows about 7% to about 15% of pharmaceutical agent iseluted one day after the device is contacted with elution media; 25% toabout 35% of pharmaceutical agent is eluted 7 days after the device iscontacted with elution media; about 35% to about 55% of pharmaceuticalagent is eluted 14 days after the device is contacted with elutionmedia; about 45% to about 60% of pharmaceutical agent is eluted 21 daysafter the device is contacted with elution media; and about 50% to about70% of pharmaceutical agent is eluted 28 days after the device iscontacted with elution media.

Provided herein is a device comprising a stent; and a plurality oflayers on said stent; wherein at least one of said layers comprises abioabsorbable polymer and at least one of said layers comprises apharmaceutical agent selected from rapamycin, a prodrug, a derivative,an analog, a hydrate, an ester, and a salt thereof, wherein said deviceprovides an in vitro pharmaceutical agent elution profile wherein saidelution profile shows at least 5% of pharmaceutical agent is eluted oneday after the device is contacted with elution media; at least 15% ofpharmaceutical agent is eluted 7 days after the device is contacted withelution media; at least 25% of pharmaceutical agent is eluted 14 daysafter the device is contacted with elution media; at least 30% ofpharmaceutical agent is eluted 21 days after the device is contactedwith elution media; at least 40% of pharmaceutical agent is eluted 28days after the device is contacted with elution media.

Provided herein is a device comprising a stent; and a plurality oflayers on said stent; wherein at least one of said layers comprises abioabsorbable polymer and at least one of said layers comprises apharmaceutical agent selected from rapamycin, a prodrug, a derivative,an analog, a hydrate, an ester, and a salt thereof, wherein said deviceprovides an in vitro pharmaceutical agent elution profile wherein saidelution profile shows about 10% of pharmaceutical agent is eluted oneday after the device is contacted with elution media; about 30% ofpharmaceutical agent is eluted 7 days after the device is contacted withelution media; about 45% of pharmaceutical agent is eluted 14 days afterthe device is contacted with elution media; about 50% of pharmaceuticalagent is eluted 21 days after the device is contacted with elutionmedia; about 60% of pharmaceutical agent is eluted 28 days after thedevice is contacted with elution media.

Provided herein is a device comprising a stent; and a plurality oflayers on said stent; wherein at least one of said layers comprises abioabsorbable polymer and at least one of said layers comprises apharmaceutical agent selected from rapamycin, a prodrug, a derivative,an analog, a hydrate, an ester, and a salt thereof, wherein said deviceprovides an in vitro pharmaceutical agent elution profile wherein saidelution profile shows about 10% to about 75% of pharmaceutical agent iseluted at week 1 after the device is contacted with elution media, about25% to about 85% of pharmaceutical agent is eluted at week 2 and about50% to about 100% of pharmaceutical agent is eluted at week 10.

Provided herein is a device comprising: a stent; and a plurality oflayers on said stent; wherein at least one of said layers comprises abioabsorbable polymer and at least one of said layers comprises apharmaceutical agent selected from rapamycin, a prodrug, a derivative,an analog, a hydrate, an ester, and a salt thereof, wherein said deviceprovides an in vitro pharmaceutical agent elution profile shown in FIG.5.

In some embodiments, the in vitro pharmaceutical agent elution profileis determined by a procedure comprising: (i) contacting the device withan elution media comprising 5% ethanol by volume wherein the pH of themedia is about 7.4 and wherein the device is contacted with the elutionmedia at a temperature of about 37° C.; (ii) optionally agitating theelution media during the contacting step in (i); (iii) removing theelution media at designated time points; and (iv) assaying the removedelution media to determine pharmaceutical agent content.

In some embodiments, the in vitro pharmaceutical agent elution profileis determined by a procedure comprising: (i) contacting the device withan elution media comprising 5% ethanol by volume, wherein the pH of themedia is about 7.4 and wherein the device is contacted with the elutionmedia at a temperature of about 37° C.; (ii) optionally agitating theelution media during the contacting step in (i); (iii) removing saiddevice from the elution media at designated time points; and (iv)assaying the elution media to determine pharmaceutical agent content.

In some embodiments, the in vitro pharmaceutical agent elution profileis determined in the absence of agitation.

In some embodiments, the procedure further comprises: (v) determiningpolymer weight loss by comparing the weight of the device before andafter the contacting step and adjusting for the amount of pharmaceuticalagent eluted into the elution media as determined in step (iv). In someembodiments, step (v) shows at least 50% of polymer is released into themedia after the device is contacted with the media for 90 days or more.In some embodiments, step (v) shows at least 75% of polymer is releasedinto the media after the device is contacted with the media for 90 daysor more.

In some embodiments, step (v) shows at least 85% of polymer is releasedinto the media after the device is contacted with the media for 90 daysor more. In some embodiments, step (v) shows at least 50% of polymer isreleased into the media after the device is contacted with the media forabout 90 days. In some embodiments, step (v) shows at least 75% ofpolymer is released into the media after the device is contacted withthe media for about 90 days. In some embodiments, step (v) shows atleast 85% of polymer is released into the media after the device iscontacted with the media for about 90 days. In some embodiments, step(v) shows at least 95% of polymer is released into the media after thedevice is contacted with the media for about 90 days. In someembodiments, step (v) shows up to 100% of polymer is released into themedia after the device is contacted with the media for about 90 days.

Provided herein is a device comprising: a stent; and a plurality oflayers on said stent; wherein at least one of said layers comprises abioabsorbable polymer and at least one of said layers comprises apharmaceutical agent selected from rapamycin, a prodrug, a derivative,an analog, a hydrate, an ester, and a salt thereof, wherein said deviceprovides an in vitro pharmaceutical agent elution profile wherein saidelution profile shows about 1% to about 35% of pharmaceutical agent iseluted one hour after the device is contacted with elution media; 5% toabout 45% of pharmaceutical agent is eluted 3 hours after the device iscontacted with elution media; about 30% to about 70% of pharmaceuticalagent is eluted 1 day after the device is contacted with elution media;about 40% to about 80% of pharmaceutical agent is eluted 3 days afterthe device is contacted with elution media; about 50% to about 90% ofpharmaceutical agent is eluted 10 days after the device is contactedwith elution media about 55% to about 95% of pharmaceutical agent iseluted 15 days after the device is contacted with elution media; andabout 60% to about 100% of pharmaceutical agent is eluted 20 days afterthe device is contacted with elution media.

Provided herein is a device comprising: a stent; and a plurality oflayers on said stent; wherein at least one of said layers comprises abioabsorbable polymer and at least one of said layers comprises apharmaceutical agent selected from rapamycin, a prodrug, a derivative,an analog, a hydrate, an ester, and a salt thereof, wherein said deviceprovides an in vitro pharmaceutical agent elution profile wherein saidelution profile shows about 5% to about 25% of pharmaceutical agent iseluted one hour after the device is contacted with elution media; 5% toabout 35% of pharmaceutical agent is eluted 3 hours after the device iscontacted with elution media; about 30% to about 65% of pharmaceuticalagent is eluted 1 day after the device is contacted with elution media;about 45% to about 70% of pharmaceutical agent is eluted 3 days afterthe device is contacted with elution media; about 55% to about 85% ofpharmaceutical agent is eluted 10 days after the device is contactedwith elution media about 65% to about 85% of pharmaceutical agent iseluted 15 days after the device is contacted with elution media; andabout 75% to about 100% of pharmaceutical agent is eluted 20 days afterthe device is contacted with elution media.

Provided herein is a device comprising: a stent; and a plurality oflayers on said stent; wherein at least one of said layers comprises abioabsorbable polymer and at least one of said layers comprises apharmaceutical agent selected from rapamycin, a prodrug, a derivative,an analog, a hydrate, an ester, and a salt thereof, wherein said deviceprovides an in vitro pharmaceutical agent elution profile shown in FIG.9.

In some embodiments, the in vitro pharmaceutical agent elution profileis determined by a procedure comprising: (i) contacting the device withan elution media comprising ethanol and phosphate buffered salinewherein the pH of the media is about 7.4 and wherein the device iscontacted with the elution media at a temperature of about 37° C.; (ii)optionally agitating the elution media during the contacting step in(i); (iii) removing the elution media at designated time points; and(iv) assaying the removed elution media to determine pharmaceuticalagent content.

In some embodiments, the in vitro pharmaceutical agent elution profileis determined by a procedure comprising: (i) contacting the device withan elution media comprising ethanol and phosphate buffered salinewherein the pH of the media is about 7.4 and wherein the device iscontacted with the elution media at a temperature of about 37° C.; (ii)optionally agitating the elution media during the contacting step in(i); (iii) removing said device from the elution media at designatedtime points; and (iv) assaying the elution media to determinepharmaceutical agent content.

In some embodiments, the in vitro pharmaceutical agent elution profileis determined in the absence of agitation.

In some embodiments, the procedure further comprises: (v) determiningpolymer weight loss by comparing the weight of the device before andafter the contacting step and adjusting for the amount of pharmaceuticalagent eluted into the elution media as determined in step iv. The deviceof claim 160 wherein step v shows at least 50% of polymer is releasedinto the media after the device is contacted with the media for 90 daysor more.

In some embodiments, step (v) shows at least 75% of polymer is releasedinto the media after the device is contacted with the media for 90 daysor more. In some embodiments, step (v) shows at least 85% of polymer isreleased into the media after the device is contacted with the media for90 days or more. In some embodiments, step (v) shows at least 50% ofpolymer is released into the media after the device is contacted withthe media for about 90 days. In some embodiments, step (v) shows atleast 75% of polymer is released into the media after the device iscontacted with the media for about 90 days. In some embodiments, step(v) shows at least 85% of polymer is released into the media after thedevice is contacted with the media for about 90 days. In someembodiments, step (v) shows at least 95% of polymer is released into themedia after the device is contacted with the media for about 90 days.

Provided herein is a device comprising: a stent; and a coatingcomprising a pharmaceutical agent selected from rapamycin, a prodrug, aderivative, ester and a salt thereof and a polymer wherein the coatinghas an initial pharmaceutical agent amount; wherein when said device isdelivered in a body lumen of a subject the pharmaceutical agent isdelivered in vessel wall tissue of the subject as follows: from about0.1% to about 35% of the initial pharmaceutical agent amount isdelivered in the subject's vessel wall tissue one week after the deviceis delivered in the subject's body; and from about 0.5% to about 50% ofthe initial pharmaceutical agent amount is delivered in the subject'svessel wall tissue two weeks after the device is delivered in thesubject's body.

In some embodiments, the amount delivered to the subject's lumen isobtained by adding pharmaceutical agent present alone in said subject'svessel wall tissue and pharmaceutical agent delivered together with saidpolymer. In some embodiments, the subject is a human.

In some embodiments, subject is a pig and the amount of pharmaceuticalagent delivered in the subject's vessel wall tissue is determined asfollows: delivering the device in the pig's blood vessel lumen;euthanizing the pig at predetermined period of time after the device isdelivered in the pig's blood vessel lumen and explanting the device;measuring the amount of pharmaceutical agent delivered in the vesselwall tissue.

Provided herein, a device comprising: a stent; and a coating comprisinga pharmaceutical agent selected from rapamycin, a prodrug, a derivative,an analog, a hydrate, an ester, and a salt thereof and a bioabsorbablepolymer wherein the coating has an initial pharmaceutical agent contentof about 1 μg/mm to about 15 μg/mm; wherein said device provides an areaunder a curve (AUC) for content of pharmaceutical agent delivered in thevessel wall tissue of a subject over time as follows: from about 0.05(μg/mm)*day to about 1 (μg/mm)*day when AUC is calculated from the timethe device is delivered in a subject's body to one day after the deviceis delivered in the subject's body; from about 5 (μg/mm)*day to about 10(μg/mm)*day when AUC is calculated starting after the first week thedevice is delivered in the subject's body through the second week afterthe device is delivered in the subject's body; from about 10 (μg/mm)*dayto about 20 (μg/mm)*day when AUC is calculated starting after the secondweek the device is delivered in the subject's body through the fourthweek after the device is delivered in the subject's body; and an AUClast of from about 40 (μg/mm)*day to about 60 (μg/mm)*day.

Provided herein is a device comprising: a stent; and a coatingcomprising a pharmaceutical agent selected from rapamycin, a prodrug, aderivative, an analog, a hydrate, an ester, and a salt thereof and abioabsorbable polymer wherein the coating has an initial polymer amount;wherein when said device is delivered in a body lumen of a subject about75% of polymer is released from the device 90 days or more after thedevice is delivered in the body lumen of the subject.

Provided herein is a device comprising: a stent; and a coatingcomprising a pharmaceutical agent selected from rapamycin, a prodrug, aderivative, an analog, a hydrate, an ester, and a salt thereof and abioabsorbable polymer wherein the coating has an initial polymer amount;wherein when said device is delivered in a body lumen of a subject about85% of polymer is released from the device about 90 days after thedevice is delivered in the body lumen of the subject.

Provided herein is a device comprising: a stent; and a coatingcomprising a pharmaceutical agent selected from rapamycin, a prodrug, aderivative, an analog, a hydrate, an ester, and a salt thereof and abioabsorbable polymer wherein the coating has an initial polymer amount;wherein when said device is delivered in a body lumen of a subject atleast about 75% of polymer is released from the device about 90 daysafter the device is delivered in the body lumen of the subject.

Provided herein is a device comprising: a stent; and a coatingcomprising a pharmaceutical agent selected from rapamycin, a prodrug, aderivative, an analog, a hydrate, an ester, and a salt thereof and abioabsorbable polymer wherein the coating has an initial polymer amount;wherein when said device is delivered in a body lumen of a subject about100% of polymer is released from the device about 90 days after thedevice is delivered in the body lumen of the subject.

In some embodiments, the subject is a human. In some embodiments, thesubject is a pig and the amount of polymer released from the device isdetermined as follows: delivering the device in the pig's blood vessellumen; euthanizing the pig at predetermined period of time after thedevice is delivered in the pig's blood vessel lumen and explanting thedevice; and measuring the amount of polymer released from the device.

In some embodiments, measuring the amount of polymer released from thedevice comprises LC/MS/MS measurements. In some embodiments, measuringthe amount released from the device comprises weight loss measurement.In some embodiments, weight loss measurement comprises measuring anamount of polymer remaining in the device and subtracting said remainingamount from the initial amount present in the device prior to deliveringthe device to the pig's blood vessel lumen.

Provided herein is a device comprising a stent; and a plurality oflayers on said stent; wherein at least one of said layers comprises abioabsorbable polymer and at least one of said layers comprises apharmaceutical agent selected from rapamycin, a prodrug, a derivative,an analog, a hydrate, an ester, and a salt thereof, wherein the devicehas an initial pharmaceutical agent content of about 1 μg/mm to about 15μg/mm; wherein when said device is delivered in a body lumen of asubject said device provides a blood concentration within 60 minutesfrom delivery of said device to the subject's body lumen that is fromabout 1% to about 50% of the blood concentration provided by aconventional drug eluting stent delivered to the subject under similarconditions.

Provided herein is a device comprising a stent; and a plurality oflayers on said stent; wherein at least one of said layers comprises abioabsorbable polymer and at least one of said layers comprises apharmaceutical agent selected from rapamycin, a prodrug, a derivative,an analog, a hydrate, an ester, and a salt thereof, wherein the devicehas an initial pharmaceutical agent content of about 1 μg/mm to about 15μg/mm; wherein when said device is delivered in a body lumen of asubject said device provides a blood concentration within 60 minutesfrom delivery of said device to the subject's body lumen that is fromabout 11% to about 20% of the blood concentration provided by aconventional drug eluting stent delivered to the subject under similarconditions.

Provided herein is a device comprising a stent; and coating on saidstent; wherein said coating comprises a bioabsorbable polymer and apharmaceutical agent selected from rapamycin, a prodrug, a derivative,an analog, a hydrate, an ester, and a salt thereof, wherein the devicehas an initial pharmaceutical agent content of about 1 μg/mm to about 15μg/mm; wherein when said device is delivered in a body lumen of asubject said device provides about the same blood concentration over thefirst 72 hours from delivery of said device to the subject's body lumen.

In some embodiments, the blood concentration during the first 72 hoursfrom delivery of said device to the subject's body lumen remains between75% and 125% of an average blood concentration calculated over the first72 hours from delivery of said device to the subject's body lumen. Insome embodiments, the average blood concentration is from about 0.05ng/mL to about 0.5 ng/mL. In some embodiments, the device provides anAUC for blood concentration over a period of 72 hours after the deviceis delivered to the subject's body lumen of from about 2 (ng/mL)*hour toabout 20 (ng/mL)*hour.

In some embodiments, the device provides an AUC for blood concentrationover a period of 72 hours after the device is delivered to the subject'sbody lumen of from about 4 (ng/mL)*hour to about 10 (ng/mL)*hour. Insome embodiments, at least part of pharmaceutical agent is incrystalline form. In some embodiments, the pharmaceutical agent isprovided at a reduced dose compared to a conventional drug elutingstent. In some embodiments, at least one of said layers comprises a PLGAbioabsorbable polymer.

In some embodiments, the pharmaceutical agent in said device has a shelfstability of at least 12 months.

In some embodiments, the device provides an in vitro pharmaceuticalagent elution profile comparable to first order kinetics.

In some embodiments, the device provides pharmaceutical agent tissueconcentration of at least twice the tissue concentration provided by aconventional stent. In some embodiments, the device provides apharmaceutical agent tissue concentration of at least 5 times greaterthan the tissue concentration provided by a conventional stent. In someembodiments, the device provides a pharmaceutical agent tissueconcentration of at least 25 times greater than the tissue concentrationprovided by a conventional stent. In some embodiments, the deviceprovides a pharmaceutical agent tissue concentration of at least 100times greater than the tissue concentration provided by a conventionalstent.

In some embodiments, about 50% of said polymer is resorbed within 45-90days after an angioplasty procedure wherein said device is delivered ina subject's body. In some embodiments, about 75% of said polymer isresorbed within 45-90 days after an angioplasty procedure wherein saiddevice is delivered in a subject's body. In some embodiments, about 95%of said polymer is resorbed within 45-90 days after an angioplastyprocedure wherein said device is delivered in a subject's body.

In some embodiments, 99% of said polymer is resorbed within 45-90 daysafter an angioplasty procedure wherein said device is delivered in asubject's body.

In some embodiments, the device provides reduced inflammation over thecourse of polymer resorbtion compared to a conventional stent.

Provided herein is a method of treating a subject comprising deliveringa device as described herein in a body lumen.

Provided herein, is a method of treating a subject comprising deliveringin the body of the subject a device comprising: a stent; and a coatingcomprising a pharmaceutical agent selected from rapamycin, a prodrug, aderivative, an analog, a hydrate, an ester, and a salt thereof and apolymer wherein the coating has an initial pharmaceutical agent amount;wherein said device is delivered in a body lumen of the subject and thepharmaceutical agent is delivered in vessel wall tissue of the subjectas follows: i. from about 0.05% to about 35% of the initialpharmaceutical agent amount is delivered in the subject's vessel walltissue one week after the device is delivered in the subject's body; andii. from about 0.5% to about 50% of the initial pharmaceutical agentamount is delivered in the subject's vessel wall tissue two weeks afterthe device is delivered in the subject's body.

In some embodiments, the device provides reduced inflammation over thecourse of polymer resorbtion.

In some embodiments, the presence of crystallinity is shown by at leastone of XRD, Raman Spectroscopy, Infrared analytical methods, and DSC.

In some embodiments, the coating on an abluminal surface of said stenthas a greater thickness than coating on a luminal surface of said stent.In some embodiments, the ratio of coating on the abluminal surface tocoating on the luminal surface of the device is 80:20. In someembodiments, the ratio of coating on the abluminal surface to coating onthe luminal surface of the device is 75:25. In some embodiments, theratio of coating on the abluminal surface to coating on the luminalsurface of the device is 70:30. In some embodiments, the ratio ofcoating on the abluminal surface to coating on the luminal surface ofthe device is 60:40.

In some embodiments, the stent is a coronary stent, a vascular stent, aperipheral stent, billiarty stent, and intercranial stent.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1: Bioabsorbability testing of 50:50 PLGA-ester end group (MW ˜19kD) polymer coating formulations on stents by determination of pHChanges with Polymer Film Degradation in 20% Ethanol/Phosphate BufferedSaline as set forth in Example 3 described herein.

FIG. 2: Bioabsorbability testing of 50:50 PLGA-carboxylate end group (MW10 kD) PLGA polymer coating formulations on stents by determination ofpH Changes with Polymer Film Degradation in 20% Ethanol/PhosphateBuffered Saline as set forth in Example 3 described herein.

FIG. 3: Bioabsorbability testing of 85:15 (85% lactic acid, 15% glycolicacid) PLGA polymer coating formulations on stents by determination of pHChanges with Polymer Film Degradation in 20% Ethanol/Phosphate BufferedSaline as set forth in Example 3 described herein.

FIG. 4: Bioabsorbability testing of various PLGA polymer coating filmformulations by determination of pH Changes with Polymer FilmDegradation in 20% Ethanol/Phosphate Buffered Saline as set forth inExample 3 described herein.

FIG. 5: Rapamycin Elution Profile of coated stents (PLGA/Rapamycincoatings) where the elution profile was determined by a static elutionmedia of 5% EtOH/water, pH 7.4, 37° C. via UV-Vis test method asdescribed in Example 11b of coated stents described therein.

FIG. 6: Rapamycin Elution Profile of coated stents (PLGA/Rapamycincoatings) where the elution profile was determined by static elutionmedia of 5% EtOH/water, pH 7.4, 37° C. via a UV-Vis test method asdescribed in Example 11b of coated stents described therein. FIG. 6depicts AS1 and AS2 as having statistically different elution profiles;AS2 and AS2b have stastically different profiles; AS1 and AS1b are notstatistically different; and AS2 and AS1(213) begin to converge at 35days. FIG. 6 suggests that the coating thickness does not affect elutionrates form 3095 polymer, but does affect elution rates from the 213polymer.

FIG. 7: Rapamycin Elution Rates of coated stents (PLGA/Rapamycincoatings) where the static elution profile was compared with agitatedelution profile by an elution media of 5% EtOH/water, pH 7.4, 37° C. viaa UV-Vis test method a UV-Vis test method as described in Example 11b ofcoated stents described therein. FIG. 7 depicts that agitation inelution media increases the rate of elution for AS2 stents, but is notstatistically significantly different for AS1 stents. The profiles arebased on two stent samples.

FIG. 8 Rapamycin Elution Profile of coated stents (PLGA/Rapamycincoatings) where the elution profile by 5% EtOH/water, pH 7.4, 37° C.elution buffer was compare with the elution profile using phosphatebuffer saline pH 7.4, 37° C.; both profiles were determined by a UV-Vistest method as described in Example 11b of coated stents describedtherein. FIG. 8 depicts that agitating the stent in elution mediaincreases the elution rate in phosphate buffered saline, but the erroris much greater.

FIG. 9: Rapamycin Elution Profile of coated stents (PLGA/Rapamycincoatings) where the elution profile was determined by a 20%EtOH/phosphate buffered saline, pH 7.4, 37° C. elution buffer and a HPLCtest method as described in Example 11c described therein, wherein theelution time (x-axis) is expressed linearly.

FIG. 10: Rapamycin Elution Profile of coated stents (PLGA/Rapamycincoatings) where the elution profile was determined by a 20%EtOH/phosphate buffered saline, pH 7.4, 37° C. elution buffer and a HPLCtest method as described in Example 11c of described therein, whereinthe elution time (x-axis) is expressed in logarithmic scale (i.e.,log(time)).

FIG. 11: Vessel wall tissue showing various elements near the lumen.

FIG. 12: Low-magnification cross-sections of porcine coronary arterystent implants (AS1, AS2 and Bare-metal stent control) at 28 dayspost-implantation as described in Example 25.

FIG. 13: Low-magnification cross-sections of porcine coronary arterystent implants (AS1, AS2 and Bare-metal stent control) at 90 dayspost-implantation as described in Example 25.

FIG. 14: Low-magnification cross-sections of porcine coronary arterystent implants depicting AS1 and AS2 drug depots as described in Example25.

FIG. 15: Low-magnification cross-sections of porcine coronary artery AS1stent implants at 90 days depicting drug depots as described in Example25.

FIG. 16: Mean (n=3) Sirolimus Levels in Arterial Tissue Following AS1and Cypher Stent Implantations in Swine Coronary Arteries expressed asabsolute tissue level (y-axis) versus time (x-axis) following testing asdescribed in Example 25. The results for AS1 presented in FIG. 16 weretaken from a separate study as the results for the Cypher Stentspresented in FIG. 16. Both studies were performed as described inExample 25, and data was collected similarly, however, data from the twostudies were combined in this Figure to show the relative results of AS1to the Cypher stents.

FIG. 17: Mean (n=3) Sirolimus Levels in Arterial Tissue FollowingVarious Stent Implantations in Swine Coronary Arteries expressed asabsolute tissue level (y-axis) versus time (x-axis) following testing asdescribed in Example 25.

FIG. 18: Arterial Tissue Concentrations (y-axis) versus time (x-axis)for AS1 and AS2 stents following testing as described in Example 25.

FIG. 19: Mean (n=3) Sirolimus Levels in Arterial Tissue FollowingVarious Stent Implantations in Swine Coronary Arteries expressed asstent level (y-axis) versus time (x-axis) following testing as describedin Example 25.

FIG. 20: Mean (n=3) Sirolimus Levels remaining on stents in FollowingAS1 and Cypher Stent Implantations in Swine Coronary Arteries expressedas stent level (y-axis) versus time (x-axis) following testing asdescribed in Example 25. The results for AS1 presented in FIG. 20 weretaken from a separate study as the results for the Cypher Stentspresented in FIG. 20. Both studies were performed as described inExample 25, and data was collected similarly, however, data from the twostudies were combined in this Figure to show the relative results of AS1to the Cypher stents.

FIG. 21: Fractional Sirolimus Release (y-axis) versus time (x-axis) inArterial Tissue for AS1 and AS2 Stents following testing as described inExample 25.

FIG. 22: Sirolimus Blood Concentration following Single Stent Implantexpressed in Blood Concentration (ng/mL) (y-axis) versus time (x-axis)following testing as described in Example 25.

FIG. 23: Mean (Single stent normalized) Blood Concentration Immediatelypost implant (between 15 minutes and 1 hour, typically 30 minutes)expressed as Blood Concentrations (ng/mL) (y-axis) for a Cypher stent,and stents having coatings as described herein (AS21, AS1, AS23, AS24are devices comprising coatings as described herein) following testingas described in Example 25.

DETAILED DESCRIPTION

The present invention is explained in greater detail below. Thisdescription is not intended to be a detailed catalog of all thedifferent ways in which the invention may be implemented, or all thefeatures that may be added to the instant invention. For example,features illustrated with respect to one embodiment may be incorporatedinto other embodiments, and features illustrated with respect to aparticular embodiment may be deleted from that embodiment. In addition,numerous variations and additions to the various embodimentscontemplated herein will be apparent to those skilled in the art inlight of the instant disclosure, which do not depart from the instantinvention. Hence, the following specification is intended to illustrateselected embodiments of the invention, and not to exhaustively specifyall permutations, combinations and variations thereof.

DEFINITIONS

As used in the present specification, the following words and phrasesare generally intended to have the meanings as set forth below, exceptto the extent that the context in which they are used indicatesotherwise.

“Substrate” as used herein, refers to any surface upon which it isdesirable to deposit a coating comprising a polymer and a pharmaceuticalor biological agent, wherein the coating process does not substantiallymodify the morphology of the pharmaceutical agent or the activity of thebiological agent. Biomedical implants are of particular interest for thepresent invention; however the present invention is not intended to berestricted to this class of substrates. Those of skill in the art willappreciate alternate substrates that could benefit from the coatingprocess described herein, such as pharmaceutical tablet cores, as partof an assay apparatus or as components in a diagnostic kit (e.g. a teststrip).

“Biomedical implant” as used herein refers to any implant for insertioninto the body of a human or animal subject, including but not limited tostents (e.g., coronary stents, vascular stents including peripheralstents and graft stents, urinary tract stents, urethral/prostaticstents, rectal stent, oesophageal stent, biliary stent, pancreaticstent), electrodes, catheters, leads, implantable pacemaker,cardioverter or defibrillator housings, joints, screws, rods, ophthalmicimplants, femoral pins, bone plates, grafts, anastomotic devices,perivascular wraps, sutures, staples, shunts for hydrocephalus, dialysisgrafts, colostomy bag attachment devices, ear drainage tubes, leads forpace makers and implantable cardioverters and defibrillators, vertebraldisks, bone pins, suture anchors, hemostatic barriers, clamps, screws,plates, clips, vascular implants, tissue adhesives and sealants, tissuescaffolds, various types of dressings (e.g., wound dressings), bonesubstitutes, intraluminal devices, vascular supports, etc.

The implants may be formed from any suitable material, including but notlimited to polymers (including stable or inert polymers, organicpolymers, organic-inorganic copolymers, inorganic polymers, andbiodegradable polymers), metals, metal alloys, inorganic materials suchas silicon, and composites thereof, including layered structures with acore of one material and one or more coatings of a different material.Substrates made of a conducting material facilitate electrostaticcapture. However, the invention contemplates the use of electrostaticcapture, as described below, in conjunction with substrate having lowconductivity or which are non-conductive. To enhance electrostaticcapture when a non-conductive substrate is employed, the substrate isprocessed for example while maintaining a strong electrical field in thevicinity of the substrate.

Subjects into which biomedical implants of the invention may be appliedor inserted include both human subjects (including male and femalesubjects and infant, juvenile, adolescent, adult and geriatric subjects)as well as animal subjects (including but not limited to pig, rabbit,mouse, dog, cat, horse, monkey, etc.) for veterinary purposes and/ormedical research.

In a preferred embodiment the biomedical implant is an expandableintraluminal vascular graft or stent (e.g., comprising a wire mesh tube)that can be expanded within a blood vessel by an angioplasty balloonassociated with a catheter to dilate and expand the lumen of a bloodvessel, such as described in U.S. Pat. No. 4,733,665 to Palmaz.

“Pharmaceutical agent” as used herein refers to any of a variety ofdrugs or pharmaceutical compounds that can be used as active agents toprevent or treat a disease (meaning any treatment of a disease in amammal, including preventing the disease, i.e. causing the clinicalsymptoms of the disease not to develop; inhibiting the disease, i.e.arresting the development of clinical symptoms; and/or relieving thedisease, i.e. causing the regression of clinical symptoms). It ispossible that the pharmaceutical agents of the invention may alsocomprise two or more drugs or pharmaceutical compounds. Pharmaceuticalagents, include but are not limited to antirestenotic agents,antidiabetics, analgesics, antiinflammatory agents, antirheumatics,antihypotensive agents, antihypertensive agents, psychoactive drugs,tranquillizers, antiemetics, muscle relaxants, glucocorticoids, agentsfor treating ulcerative colitis or Crohn's disease, antiallergics,antibiotics, antiepileptics, anticoagulants, antimycotics, antitussives,arteriosclerosis remedies, diuretics, proteins, peptides, enzymes,enzyme inhibitors, gout remedies, hormones and inhibitors thereof,cardiac glycosides, immunotherapeutic agents and cytokines, laxatives,lipid-lowering agents, migraine remedies, mineral products, otologicals,anti parkinson agents, thyroid therapeutic agents, spasmolytics,platelet aggregation inhibitors, vitamins, cytostatics and metastasisinhibitors, phytopharmaceuticals, chemotherapeutic agents and aminoacids. Examples of suitable active ingredients are acarbose, antigens,beta-receptor blockers, non-steroidal antiinflammatory drugs [NSAIDs],cardiac glycosides, acetylsalicylic acid, virustatics, aclarubicin,acyclovir, cisplatin, actinomycin, alpha- and beta-sympatomimetics,(dmeprazole, allopurinol, alprostadil, prostaglandins, amantadine,ambroxol, amlodipine, methotrexate, S-aminosalicylic acid,amitriptyline, amoxicillin, anastrozole, atenolol, azathioprine,balsalazide, beclomethasone, betahistine, bezafibrate, bicalutamide,diazepam and diazepam derivatives, budesonide, bufexamac, buprenorphine,methadone, calcium salts, potassium salts, magnesium salts, candesartan,carbamazepine, captopril, cefalosporins, cetirizine, chenodeoxycholicacid, ursodeoxycholic acid, theophylline and theophylline derivatives,trypsins, cimetidine, clarithromycin, clavulanic acid, clindamycin,clobutinol, clonidine, cotrimoxazole, codeine, caffeine, vitamin D andderivatives of vitamin D, colestyramine, cromoglicic acid, coumarin andcoumarin derivatives, cysteine, cytarabine, cyclophosphamide,ciclosporin, cyproterone, cytabarine, dapiprazole, desogestrel,desonide, dihydralazine, diltiazem, ergot alkaloids, dimenhydrinate,dimethyl sulphoxide, dimeticone, domperidone and domperidan derivatives,dopamine, doxazosin, doxorubizin, doxylamine, dapiprazole,benzodiazepines, diclofenac, glycoside antibiotics, desipramine,econazole, ACE inhibitors, enalapril, ephedrine, epinephrine, epoetinand epoetin derivatives, morphinans, calcium antagonists, irinotecan,modafinil, orlistat, peptide antibiotics, phenyloin, riluzoles,risedronate, sildenafil, topiramate, macrolide antibiotics, oestrogenand oestrogen derivatives, progestogen and progestogen derivatives,testosterone and testosterone derivatives, androgen and androgenderivatives, ethenzamide, etofenamate, etofibrate, fenofibrate,etofylline, etoposide, famciclovir, famotidine, felodipine, fenofibrate,fentanyl, fenticonazole, gyrase inhibitors, fluconazole, fludarabine,fluarizine, fluorouracil, fluoxetine, flurbiprofen, ibuprofen,flutamide, fluvastatin, follitropin, formoterol, fosfomicin, furosemide,fusidic acid, gallopamil, ganciclovir, gemfibrozil, gentamicin, ginkgo,Saint John's wort, glibenclamide, urea derivatives as oralantidiabetics, glucagon, glucosamine and glucosamine derivatives,glutathione, glycerol and glycerol derivatives, hypothalamus hormones,goserelin, gyrase inhibitors, guanethidine, halofantrine, haloperidol,heparin and heparin derivatives, hyaluronic acid, hydralazine,hydrochlorothiazide and hydrochlorothiazide derivatives, salicylates,hydroxyzine, idarubicin, ifosfamide, imipramine, indometacin,indoramine, insulin, interferons, iodine and iodine derivatives,isoconazole, isoprenaline, glucitol and glucitol derivatives,itraconazole, ketoconazole, ketoprofen, ketotifen, lacidipine,lansoprazole, levodopa, levomethadone, thyroid hormones, lipoic acid andlipoic acid derivatives, lisinopril, lisuride, lofepramine, lomustine,loperamide, loratadine, maprotiline, mebendazole, mebeverine, meclozine,mefenamic acid, mefloquine, meloxicam, mepindolol, meprobamate,meropenem, mesalazine, mesuximide, metamizole, metformin, methotrexate,methylphenidate, methylprednisolone, metixene, metoclopramide,metoprolol, metronidazole, mianserin, miconazole, minocycline,minoxidil, misoprostol, mitomycin, mizolastine, moexipril, morphine andmorphine derivatives, evening primrose, nalbuphine, naloxone, tilidine,naproxen, narcotine, natamycin, neostigmine, nicergoline, nicethamide,nifedipine, niflumic acid, nimodipine, nimorazole, nimustine,nisoldipine, adrenaline and adrenaline derivatives, norfloxacin,novamine sulfone, noscapine, nystatin, ofloxacin, olanzapine,olsalazine, omeprazole, omoconazole, ondansetron, oxaceprol, oxacillin,oxiconazole, oxymetazoline, pantoprazole, paracetamol, paroxetine,penciclovir, oral penicillins, pentazocine, pentifylline,pentoxifylline, perphenazine, pethidine, plant extracts, phenazone,pheniramine, barbituric acid derivatives, phenylbutazone, phenyloin,pimozide, pindolol, piperazine, piracetam, pirenzepine, piribedil,piroxicam, pramipexole, pravastatin, prazosin, procaine, promazine,propiverine, propranolol, propyphenazone, prostaglandins, protionamide,proxyphylline, quetiapine, quinapril, quinaprilat, ramipril, ranitidine,reproterol, reserpine, ribavirin, rifampicin, risperidone, ritonavir,ropinirole, roxatidine, roxithromycin, ruscogenin, rutoside and rutosidederivatives, sabadilla, salbutamol, salmeterol, scopolamine, selegiline,sertaconazole, sertindole, sertralion, silicates, sildenafil,simvastatin, sitosterol, sotalol, spaglumic acid, sparfloxacin,spectinomycin, spiramycin, spirapril, spironolactone, stavudine,streptomycin, sucralfate, sufentanil, sulbactam, sulphonamides,sulfasalazine, sulpiride, sultamicillin, sultiam, sumatriptan,suxamethonium chloride, tacrine, tacrolimus, taliolol, tamoxifen,taurolidine, tazarotene, temazepam, teniposide, tenoxicam, terazosin,terbinafine, terbutaline, terfenadine, terlipressin, tertatolol,tetracyclins, teryzoline, theobromine, theophylline, butizine,thiamazole, phenothiazines, thiotepa, tiagabine, tiapride, propionicacid derivatives, ticlopidine, timolol, timidazole, tioconazole,tioguanine, tioxolone, tiropramide, tizanidine, tolazoline, tolbutamide,tolcapone, tolnaftate, tolperisone, topotecan, torasemide,antioestrogens, tramadol, tramazoline, trandolapril, tranylcypromine,trapidil, trazodone, triamcinolone and triamcinolone derivatives,triamterene, trifluperidol, trifluridine, trimethoprim, trimipramine,tripelennamine, triprolidine, trifosfamide, tromantadine, trometamol,tropalpin, troxerutine, tulobuterol, tyramine, tyrothricin, urapidil,ursodeoxycholic acid, chenodeoxycholic acid, valaciclovir, valproicacid, vancomycin, vecuronium chloride, Viagra, venlafaxine, verapamil,vidarabine, vigabatrin, viloazine, vinblastine, vincamine, vincristine,vindesine, vinorelbine, vinpocetine, viquidil, warfarin, xantinolnicotinate, xipamide, zafirlukast, zalcitabine, zidovudine,zolmitriptan, zolpidem, zoplicone, zotipine and the like. See, e.g.,U.S. Pat. No. 6,897,205; see also U.S. Pat. No. 6,838,528; U.S. Pat. No.6,497,729.

Examples of therapeutic agents employed in conjunction with theinvention include, rapamycin, 40-O-(2-Hydroxyethyl)rapamycin(everolimus), 40-O-Benzyl-rapamycin,40-O-(4′-Hydroxymethyl)benzyl-rapamycin,40-O-[4′-(1,2-Dihydroxyethyl)]benzyl-rapamycin, 40-O-Allyl-rapamycin,40-O-[3′-(2,2-Dimethyl-1,3-dioxolan-4(S)-yl)-prop-2′-en-1′-yl]-rapamycin,(2′:E,4′S)-40-O-(4′,5′-Dihydroxypent-2′-en-1′-yl)-rapamycin40-O-(2-Hydroxy)ethoxycar-bonylmethyl-rapamycin,40-O-(3-Hydroxy)propyl-rapamycin 4O—O-(6-Hydroxy)hexyl-rapamycin40-O-[2-(2-Hydroxy)ethoxy]ethyl-rapamycin4O—O-[(3S)-2,2-Dimethyldioxolan-3-yl]methyl-rapamycin,40-O-[(2S)-2,3-Dihydroxyprop-1-yl]-rapamycin,4O—O-(2-Acetoxy)ethyl-rapamycin 4O—O-(2-Nicotinoyloxy)ethyl-rapamycin,4O—O-[2-(N-Morpholino)acetoxy]ethyl-rapamycin4O—O-(2-N-Imidazolylacetoxy)ethyl-rapamycin,4O—O-[2-(N-Methyl-N′-piperazinyl)acetoxy]ethyl-rapamycin,39-O-Desmethyl-39,40-O,O-ethylene-rapamycin,(26R)-26-Dihydro-40-O-(2-hydroxy)ethyl-rapamycin, 28-O-Methyl-rapamycin,4O—O-(2-Aminoethyl)-rapamycin, 4O—O-(2-Acetaminoethyl)-rapamycin4O—O-(2-Nicotinamidoethyl)-rapamycin,4O—O-(2-(N-Methyl-imidazo-2′-ylcarbethoxamido)ethyl)-rapamycin,4O—O-(2-Ethoxycarbonylaminoethyl)-rapamycin,40-O-(2-Tolylsulfonamidoethyl)-rapamycin,40-O-[2-(4′,5′-Dicarboethoxy-1′,2′,3′-triazol-1′-yl)-ethyl]-rapamycin,42-Epi-(tetrazolyl)rapamycin (tacrolimus), and42-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]rapamycin(temsirolimus).

The pharmaceutical agents may, if desired, also be used in the form oftheir pharmaceutically acceptable salts or derivatives (meaning saltswhich retain the biological effectiveness and properties of thecompounds of this invention and which are not biologically or otherwiseundesirable), and in the case of chiral active ingredients it ispossible to employ both optically active isomers and racemates ormixtures of diastereoisomers. As well, the pharmaceutical agent mayinclude a prodrug, a hydrate, an ester, a derivative or analogs of acompound or molecule.

A “pharmaceutically acceptable salt” may be prepared for anypharmaceutical agent having a functionality capable of forming a salt,for example an acid or base functionality. Pharmaceutically acceptablesalts may be derived from organic or inorganic acids and bases. The term“pharmaceutically-acceptable salts” in these instances refers to therelatively non-toxic, inorganic and organic base addition salts of thepharmaceutical agents.

“Prodrugs” are derivative compounds derivatized by the addition of agroup that endows greater solubility to the compound desired to bedelivered. Once in the body, the prodrug is typically acted upon by anenzyme, e.g., an esterase, amidase, or phosphatase, to generate theactive compound.

“Stability” as used herein in refers to the stability of the drug in apolymer coating deposited on a substrate in its final product form(e.g., stability of the drug in a coated stent). The term stability willdefine 5% or less degradation of the drug in the final product form.

“Active biological agent” as used herein refers to a substance,originally produced by living organisms, that can be used to prevent ortreat a disease (meaning any treatment of a disease in a mammal,including preventing the disease, i.e. causing the clinical symptoms ofthe disease not to develop; inhibiting the disease, i.e. arresting thedevelopment of clinical symptoms; and/or relieving the disease, i.e.causing the regression of clinical symptoms). It is possible that theactive biological agents of the invention may also comprise two or moreactive biological agents or an active biological agent combined with apharmaceutical agent, a stabilizing agent or chemical or biologicalentity. Although the active biological agent may have been originallyproduced by living organisms, those of the present invention may alsohave been synthetically prepared, or by methods combining biologicalisolation and synthetic modification. By way of a non-limiting example,a nucleic acid could be isolated form from a biological source, orprepared by traditional techniques, known to those skilled in the art ofnucleic acid synthesis. Furthermore, the nucleic acid may be furthermodified to contain non-naturally occurring moieties. Non-limitingexamples of active biological agents include peptides, proteins,enzymes, glycoproteins, nucleic acids (including deoxyribonucleotide orribonucleotide polymers in either single or double stranded form, andunless otherwise limited, encompasses known analogues of naturalnucleotides that hybridize to nucleic acids in a manner similar tonaturally occurring nucleotides), antisense nucleic acids, fatty acids,antimicrobials, vitamins, hormones, steroids, lipids, polysaccharides,carbohydrates and the like. They further include, but are not limitedto, antirestenotic agents, antidiabetics, analgesics, antiinflammatoryagents, antirheumatics, antihypotensive agents, antihypertensive agents,psychoactive drugs, tranquillizers, antiemetics, muscle relaxants,glucocorticoids, agents for treating ulcerative colitis or Crohn'sdisease, antiallergics, antibiotics, antiepileptics, anticoagulants,antimycotics, antitussives, arteriosclerosis remedies, diuretics,proteins, peptides, enzymes, enzyme inhibitors, gout remedies, hormonesand inhibitors thereof, cardiac glycosides, immunotherapeutic agents andcytokines, laxatives, lipid-lowering agents, migraine remedies, mineralproducts, otologicals, anti parkinson agents, thyroid therapeuticagents, spasmolytics, platelet aggregation inhibitors, vitamins,cytostatics and metastasis inhibitors, phytopharmaceuticals andchemotherapeutic agents. Preferably, the active biological agent is apeptide, protein or enzyme, including derivatives and analogs of naturalpeptides, proteins and enzymes. The active biological agent may also bea hormone, gene therapies, RNA, siRNA, and/or cellular therapies (fornon-limiting example, stem cells or T-cells).

“Active agent” as used herein refers to any pharmaceutical agent oractive biological agent as described herein.

“Activity” as used herein refers to the ability of a pharmaceutical oractive biological agent to prevent or treat a disease (meaning anytreatment of a disease in a mammal, including preventing the disease,i.e. causing the clinical symptoms of the disease not to develop;inhibiting the disease, i.e. arresting the development of clinicalsymptoms; and/or relieving the disease, i.e. causing the regression ofclinical symptoms). Thus the activity of a pharmaceutical or activebiological agent should be of therapeutic or prophylactic value.

“Secondary, tertiary and quaternary structure” as used herein aredefined as follows. The active biological agents of the presentinvention will typically possess some degree of secondary, tertiaryand/or quaternary structure, upon which the activity of the agentdepends. As an illustrative, non-limiting example, proteins possesssecondary, tertiary and quaternary structure. Secondary structure refersto the spatial arrangement of amino acid residues that are near oneanother in the linear sequence. The α-helix and the β-strand areelements of secondary structure. Tertiary structure refers to thespatial arrangement of amino acid residues that are far apart in thelinear sequence and to the pattern of disulfide bonds. Proteinscontaining more than one polypeptide chain exhibit an additional levelof structural organization. Each polypeptide chain in such a protein iscalled a subunit. Quaternary structure refers to the spatial arrangementof subunits and the nature of their contacts. For example hemoglobinconsists of two α and two β chains. It is well known that proteinfunction arises from its conformation or three dimensional arrangementof atoms (a stretched out polypeptide chain is devoid of activity). Thusone aspect of the present invention is to manipulate active biologicalagents, while being careful to maintain their conformation, so as not tolose their therapeutic activity.

“Polymer” as used herein, refers to a series of repeating monomericunits that have been cross-linked or polymerized. Any suitable polymercan be used to carry out the present invention. It is possible that thepolymers of the invention may also comprise two, three, four or moredifferent polymers. In some embodiments, of the invention only onepolymer is used. In some preferred embodiments a combination of twopolymers are used. Combinations of polymers can be in varying ratios, toprovide coatings with differing properties. Those of skill in the art ofpolymer chemistry will be familiar with the different properties ofpolymeric compounds.

“Copolymer” as used herein refers to a polymer being composed of two ormore different monomers. A copolymer may also and/or alternatively referto random, block, graft, copolymers known to those of skill in the art.

“Biocompatible” as used herein, refers to any material that does notcause injury or death to the animal or induce an adverse reaction in ananimal when placed in intimate contact with the animal's tissues.Adverse reactions include for example inflammation, infection, fibrotictissue formation, cell death, or thrombosis. The terms “biocompatible”and “biocompatibility” when used herein are art-recognized and mean thatthe referent is neither itself toxic to a host (e.g., an animal orhuman), nor degrades (if it degrades) at a rate that produces byproducts(e.g., monomeric or oligomeric subunits or other byproducts) at toxicconcentrations, causes inflammation or irritation, or induces an immunereaction in the host. It is not necessary that any subject compositionhave a purity of 100% to be deemed biocompatible. Hence, a subjectcomposition may comprise 99%, 98%, 97%, 96%, 95%, 90% 85%, 80%, 75% oreven less of biocompatible agents, e.g., including polymers and othermaterials and excipients described herein, and still be biocompatible.

To determine whether a polymer or other material is biocompatible, itmay be necessary to conduct a toxicity analysis. Such assays are wellknown in the art. One example of such an assay may be performed withlive carcinoma cells, such as GT3TKB tumor cells, in the followingmanner: the sample is degraded in 1 M NaOH at 37 degrees C. untilcomplete degradation is observed. The solution is then neutralized with1 M HCl. About 200 microliters of various concentrations of the degradedsample products are placed in 96-well tissue culture plates and seededwith human gastric carcinoma cells (GT3TKB) at 104/well density. Thedegraded sample products are incubated with the GT3TKB cells for 48hours. The results of the assay may be plotted as % relative growth vs.concentration of degraded sample in the tissue-culture well. Inaddition, polymers and formulations of the present invention may also beevaluated by well-known in vivo tests, such as subcutaneousimplantations in rats to confirm that they do not cause significantlevels of irritation or inflammation at the subcutaneous implantationsites.

The terms “bioabsorbable,” “biodegradable,” “bioerodible,” and“bioresorbable,” are art-recognized synonyms. These terms are usedherein interchangeably. Bioabsorbable polymers typically differ fromnon-bioabsorbable polymers in that the former may be absorbed (e.g.;degraded) during use. In certain embodiments, such use involves in vivouse, such as in vivo therapy, and in other certain embodiments, such useinvolves in vitro use. In general, degradation attributable tobiodegradability involves the degradation of a bioabsorbable polymerinto its component subunits, or digestion, e.g., by a biochemicalprocess, of the polymer into smaller, non-polymeric subunits. In certainembodiments, biodegradation may occur by enzymatic mediation,degradation in the presence of water (hydrolysis) and/or other chemicalspecies in the body, or both. The bioabsorbabilty of a polymer may beshown in-vitro as described herein or by methods known to one of skillin the art. An in-vitro test for bioabsorbability of a polymer does notrequire living cells or other biologic materials to show bioabsorptionproperties (e.g. degradation, digestion). Thus, resorbtion, resorption,absorption, absorbtion, erosion may also be used synonymously with theterms “bioabsorbable,” “biodegradable,” “bioerodible,” and“bioresorbable.” Mechanisms of degradation of a bioaborbable polymer mayinclude, but are not limited to, bulk degradation, surface erosion, andcombinations thereof.

As used herein, the term “biodegradation” encompasses both general typesof biodegradation. The degradation rate of a biodegradable polymer oftendepends in part on a variety of factors, including the chemical identityof the linkage responsible for any degradation, the molecular weight,crystallinity, biostability, and degree of cross-linking of suchpolymer, the physical characteristics (e.g., shape and size) of theimplant, and the mode and location of administration. For example, thegreater the molecular weight, the higher the degree of crystallinity,and/or the greater the biostability, the biodegradation of anybioabsorbable polymer is usually slower.

“Therapeutically desirable morphology” as used herein refers to thegross form and structure of the pharmaceutical agent, once deposited onthe substrate, so as to provide for optimal conditions of ex vivostorage, in vivo preservation and/or in vivo release. Such optimalconditions may include, but are not limited to increased shelf life,increased in vivo stability, good biocompatibility, good bioavailabilityor modified release rates. Typically, for the present invention, thedesired morphology of a pharmaceutical agent would be crystalline orsemi-crystalline or amorphous, although this may vary widely dependingon many factors including, but not limited to, the nature of thepharmaceutical agent, the disease to be treated/prevented, the intendedstorage conditions for the substrate prior to use or the location withinthe body of any biomedical implant. Preferably at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90% or 100% of the pharmaceutical agent is incrystalline or semi-crystalline form.

“Stabilizing agent” as used herein refers to any substance thatmaintains or enhances the stability of the biological agent. Ideallythese stabilizing agents are classified as Generally Regarded As Safe(GRAS) materials by the US Food and Drug Administration (FDA). Examplesof stabilizing agents include, but are not limited to carrier proteins,such as albumin, gelatin, metals or inorganic salts. Pharmaceuticallyacceptable excipient that may be present can further be found in therelevant literature, for example in the Handbook of PharmaceuticalAdditives An International Guide to More Than 6000 Products by TradeName, Chemical, Function, and Manufacturer; Michael and Irene Ash(Eds.); Gower Publishing Ltd.; Aldershot, Hampshire, England, 1995.

“Compressed fluid” as used herein refers to a fluid of appreciabledensity (e.g., >0.2 g/cc) that is a gas at standard temperature andpressure. “Supercritical fluid”, “near-critical fluid”,“near-supercritical fluid”, “critical fluid”, “densified fluid” or“densified gas” as used herein refers to a compressed fluid underconditions wherein the temperature is at least 80% of the criticaltemperature of the fluid and the pressure is at least 50% of thecritical pressure of the fluid, and/or a density of +50% of the criticaldensity of the fluid.

Examples of substances that demonstrate supercritical or near criticalbehavior suitable for the present invention include, but are not limitedto carbon dioxide, isobutylene, ammonia, water, methanol, ethanol,ethane, propane, butane, pentane, dimethyl ether, xenon, sulfurhexafluoride, halogenated and partially halogenated materials such aschlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons,perfluorocarbons (such as perfluoromethane and perfluoropropane,chloroform, trichloro-fluoromethane, dichloro-difluoromethane,dichloro-tetrafluoroethane) and mixtures thereof. Preferably, thesupercritical fluid is hexafluoropropane (FC-236EA), or1,1,1,2,3,3-hexafluoropropane. Preferably, the supercritical fluid ishexafluoropropane (FC-236EA), or 1,1,1,2,3,3-hexafluoropropane for usein PLGA polymer coatings.

“Sintering” as used herein refers to the process by which parts of thepolymer or the entire polymer becomes continuous (e.g., formation of acontinuous polymer film). As discussed below, the sintering process iscontrolled to produce a fully conformal continuous polymer (completesintering) or to produce regions or domains of continuous coating whileproducing voids (discontinuities) in the polymer. As well, the sinteringprocess is controlled such that some phase separation is obtained ormaintained between polymer different polymers (e.g., polymers A and B)and/or to produce phase separation between discrete polymer particles.Through the sintering process, the adhesions properties of the coatingare improved to reduce flaking of detachment of the coating from thesubstrate during manipulation in use. As described below, in someembodiments, the sintering process is controlled to provide incompletesintering of the polymer. In embodiments involving incomplete sintering,a polymer is formed with continuous domains, and voids, gaps, cavities,pores, channels or, interstices that provide space for sequestering atherapeutic agent which is released under controlled conditions.Depending on the nature of the polymer, the size of polymer particlesand/or other polymer properties, a compressed gas, a densified gas, anear critical fluid or a super-critical fluid may be employed. In oneexample, carbon dioxide is used to treat a substrate that has beencoated with a polymer and a drug, using dry powder and RESSelectrostatic coating processes. In another example, isobutylene isemployed in the sintering process. In other examples a mixture of carbondioxide and isobutylene is employed. In another example,1,1,2,3,3-hexafluoropropane is employed in the sintering process.

When an amorphous material is heated to a temperature above its glasstransition temperature, or when a crystalline material is heated to atemperature above a phase transition temperature, the moleculescomprising the material are more mobile, which in turn means that theyare more active and thus more prone to reactions such as oxidation.However, when an amorphous material is maintained at a temperature belowits glass transition temperature, its molecules are substantiallyimmobilized and thus less prone to reactions. Likewise, when acrystalline material is maintained at a temperature below its phasetransition temperature, its molecules are substantially immobilized andthus less prone to reactions. Accordingly, processing drug components atmild conditions, such as the deposition and sintering conditionsdescribed herein, minimizes cross-reactions and degradation of the drugcomponent. One type of reaction that is minimized by the processes ofthe invention relates to the ability to avoid conventional solventswhich in turn minimizes oxidation of drug, whether in amorphous,semi-crystalline, or crystalline form, by reducing exposure thereof tofree radicals, residual solvents, protic materials, polar-proticmaterials, oxidation initiators, and autoxidation initiators.

“Rapid Expansion of Supercritical Solutions” or “RESS” as used hereininvolves the dissolution of a polymer into a compressed fluid, typicallya supercritical fluid, followed by rapid expansion into a chamber atlower pressure, typically near atmospheric conditions. The rapidexpansion of the supercritical fluid solution through a small opening,with its accompanying decrease in density, reduces the dissolutioncapacity of the fluid and results in the nucleation and growth ofpolymer particles. The atmosphere of the chamber is maintained in anelectrically neutral state by maintaining an isolating “cloud” of gas inthe chamber. Carbon dioxide, nitrogen, argon, helium, or otherappropriate gas is employed to prevent electrical charge is transferredfrom the substrate to the surrounding environment.

“Bulk properties” properties of a coating including a pharmaceutical ora biological agent that can be enhanced through the methods of theinvention include for example: adhesion, smoothness, conformality,thickness, and compositional mixing.

“Electrostatically charged” or “electrical potential” or “electrostaticcapture” as used herein refers to the collection of the spray-producedparticles upon a substrate that has a different electrostatic potentialthan the sprayed particles. Thus, the substrate is at an attractiveelectronic potential with respect to the particles exiting, whichresults in the capture of the particles upon the substrate. i.e. thesubstrate and particles are oppositely charged, and the particlestransport through the gaseous medium of the capture vessel onto thesurface of the substrate is enhanced via electrostatic attraction. Thismay be achieved by charging the particles and grounding the substrate orconversely charging the substrate and grounding the particles, bycharging the particles at one potential (e.g. negative charge) andcharging the substrate at an opposited potential (e.g. positive charge),or by some other process, which would be easily envisaged by one ofskill in the art of electrostatic capture.

“Intimate mixture” as used herein, refers to two or more materials,compounds, or substances that are uniformly distributed or dispersedtogether.

“Layer” as used herein refers to a material covering a surface orforming an overlying part or segment. Two different layers may haveoverlapping portions whereby material from one layer may be in contactwith material from another layer. Contact between materials of differentlayers can be measured by determining a distance between the materials.For example, Raman spectroscopy may be employed in identifying materialsfrom two layers present in close proximity to each other.

While layers defined by uniform thickness and/or regular shape arecontemplated herein, several embodiments described below relate tolayers having varying thickness and/or irregular shape. Material of onelayer may extend into the space largely occupied by material of anotherlayer. For example, in a coating having three layers formed in sequenceas a first polymer layer, a pharmaceutical agent layer and a secondpolymer layer, material from the second polymer layer which is depositedlast in this sequence may extend into the space largely occupied bymaterial of the pharmaceutical agent layer whereby material from thesecond polymer layer may have contact with material from thepharmaceutical layer. It is also contemplated that material from thesecond polymer layer may extend through the entire layer largelyoccupied by pharmaceutical agent and contact material from the firstpolymer layer.

It should be noted however that contact between material from the secondpolymer layer (or the first polymer layer) and material from thepharmaceutical agent layer (e.g.; a pharmaceutical agent crystalparticle or a portion thereof) does not necessarily imply formation of amixture between the material from the first or second polymer layers andmaterial from the pharmaceutical agent layer. In some embodiments, alayer may be defined by the physical three-dimensional space occupied bycrystalline particles of a pharmaceutical agent (and/or biologicalagent). It is contemplated that such layer may or may not be continuousas phhysical space occupied by the crystal particles of pharmaceuticalagents may be interrupted, for example, by polymer material from anadjacent polymer layer. An adjacent polymer layer may be a layer that isin physical proximity to be pharmaceutical agent particles in thepharmaceutical agent layer. Similarly, an adjacent layer may be thelayer formed in a process step right before or right after the processstep in which pharmaceutical agent particles are deposited to form thepharmaceutical agent layer.

As described below, material deposition and layer formation providedherein are advantageous in that the pharmaceutical agent remains largelyin crystalline form during the entire process. While the polymerparticles and the pharmaceutical agent particles may be in contact, thelayer formation process is controlled to avoid formation of a mixturebetween the pharmaceutical agent particles the polymer particles duringformation of a coated device.

“Laminate coating” as used herein refers to a coating made up of two ormore layers of material. Means for creating a laminate coating asdescribed herein (e.g.; a laminate coating comprising bioabsorbablepolymer(s) and pharmaceutical agent) may include coating the stent withdrug and polymer as described herein (e-RESS, e-DPC, compressed-gassintering). The process comprises performing multiple and sequentialcoating steps (with sintering steps for polymer materials) whereindifferent materials may be deposited in each step, thus creating alaminated structure with a multitude of layers (at least 2 layers)including polymer layers and pharmaceutical agent layers to build thefinal device (e.g.; laminate coated stent).

The coating methods provided herein may be calibrated to provide acoating bias whereby the mount of polymer and pharmaceutical agentdeposited in the abluminal surface of the stent (exterior surface of thestent) is greater than the amount of pharmaceutical agent and amount ofpolymer deposited on the luminal surface of the stent (interior surfaceof the stent). The resulting configuration may be desirable to providepreferential elution of the drug toward the vessel wall (luminal surfaceof the stent) where the therapeutic effect of anti-restenosis isdesired, without providing the same antiproliferative drug(s) on theabluminal surface, where they may retard healing, which in turn issuspected to be a cause of late-stage safety problems with current DESs.

As well, the methods described herein provide a device wherein thecoating on the stent is biased in favor of increased coating at the endsof the stent. For example, a stent having three portions along thelength of the stent (e.g.; a central portion flanked by two endportions) may have end portions coated with increased amounts ofpharmaceutical agent and/or polymer compared to the central portion.

The present invention provides numerous advantages. The invention isadvantageous in that it allows for employing a platform combining layerformation methods based on compressed fluid technologies; electrostaticcapture and sintering methods. The platform results in drug elutingstents having enhanced therapeutic and mechanical properties. Theinvention is particularly advantageous in that it employs optimizedlaminate polymer technology. In particular, the present invention allowsthe formation of discrete layers of specific drug platforms. Asindicated above, the shape of a discrete layer of crystal particles maybe irregular, including interruptions of said layer by material fromanother layer (polymer layer) positioned in space between crystallineparticles of pharmaceutical agent.

Conventional processes for spray coating stents require that drug andpolymer be dissolved in solvent or mutual solvent before spray coatingcan occur. The platform provided herein the drugs and polymers arecoated on the stent framework in discrete steps, which can be carriedout simultaneously or alternately. This allows discrete deposition ofthe active agent (e.g., a drug) within a polymer thereby allowing theplacement of more than one drug on a single medical device with orwithout an intervening polymer layer. For example, the present platformprovides a dual drug eluting stent.

Some of the advantages provided by the subject invention includeemploying compressed fluids (e.g., supercritical fluids, for exampleE-RESS based methods); solvent free deposition methodology; a platformthat allows processing at lower temperatures thereby preserving thequalities of the active agent and the polymer; the ability toincorporate two, three or more drugs while minimizing deleteriouseffects from direct interactions between the various drugs and/or theirexcipients during the fabrication and/or storage of the drug elutingstents; a dry deposition; enhanced adhesion and mechanical properties ofthe layers on the stent framework; precision deposition and rapid batchprocessing; and ability to form intricate structures.

In one embodiment, the present invention provides a multi-drug deliveryplatform which produces strong, resilient and flexible drug elutingstents including an anti-restenosis drug (e.g., a limus or taxol) andanti-thrombosis drug (e.g., heparin or an analog thereof) and wellcharacterized bioabsorbable polymers. The drug eluting stents providedherein minimize potential for thrombosis, in part, by reducing ortotally eliminating thrombogenic polymers and reducing or totallyeliminating residual drugs that could inhibit healing.

The platform provides optimized delivery of multiple drug therapies forexample for early stage treatment (restenosis) and late-stage(thrombosis).

The platform also provides an adherent coating which enables accessthrough tortuous lesions without the risk of the coating beingcompromised.

Another advantage of the present platform is the ability to providehighly desirable eluting profiles.

Advantages of the invention include the ability to reduce or completelyeliminate potentially thrombogenic polymers as well as possibly residualdrugs that may inhibit long term healing. As well, the inventionprovides advantageous stents having optimized strength and resilience ifcoatings which in turn allows access to complex lesions and reduces orcompletely eliminates delamination. Laminated layers of bioabsorbablepolymers allow controlled elution of one or more drugs.

The platform provided herein reduces or completely eliminatesshortcoming that have been associated with conventional drug elutingstents. For example, the platform provided herein allows for much bettertuning of the period of time for the active agent to elute and theperiod of time necessary for the polymer to resorb thereby minimizingthrombosis and other deleterious effects associate with poorlycontrolled drug release.

The present invention provides several advantages which overcome orattenuate the limitations of current technology for bioabsorbablestents. Fro example, an inherent limitation of conventionalbioabsorbable polymeric materials relates to the difficulty in formingto a strong, flexible, deformable (e.g. balloon deployable) stent withlow profile. The polymers generally lack the strength ofhigh-performance metals. The present invention overcomes theselimitations by creating a laminate structure in the essentiallypolymeric stent. Without wishing to be bound by any specific theory oranalogy, the increased strength provided by the stents of the inventioncan be understood by comparing the strength of plywood vs. the strengthof a thin sheet of wood.

Embodiments of the invention involving a thin metallic stent-frameworkprovide advantages including the ability to overcome the inherentelasticity of most polymers. It is generally difficult to obtain a highrate (e.g., 100%) of plastic deformation in polymers (compared toelastic deformation where the materials have some ‘spring back’ to theoriginal shape). Again, without wishing to be bound by any theory, thecentral metal stent framework (that would be too small and weak to serveas a stent itself) would act like wires inside of a plastic, deformablestent, basically overcoming any ‘elastic memory’ of the polymer.

Another advantage of the present invention is the ability to create astent with a controlled (dialed-in) drug-elution profile. Via theability to have different materials in each layer of the laminatestructure and the ability to control the location of drug(s)independently in these layers, the method enables a stent that couldrelease drugs at very specific elution profiles, programmed sequentialand/or parallel elution profiles. Also, the present invention allowscontrolled elution of one drug without affecting the elution of a seconddrug (or different doses of the same drug).

Provided herein is a device comprising a stent; and a plurality oflayers that form a laminate coating on said stent; wherein at least oneof said layers comprises a bioabsorbable polymer and at least one ofsaid layers comprises one or more active agents; wherein at least aportion of the active agent is in crystalline form.

Provided herein is a device comprising a stent; and a plurality oflayers that form a laminate coating on said stent; wherein at least oneof said layers comprises a bioabsorbable polymer and at least one ofsaid layers comprises a pharmaceutical agent selected from rapamycin, aprodrug, a derivative, an analog, a hydrate, an ester, and a saltthereof, wherein at least a portion of the pharmaceutical agent is incrystalline form.

In some embodiments, the device has at least one pharmaceutical agentlayer defined by a three-dimensional physical space occupied by crystalparticles of said pharmaceutical agent and said three dimensionalphysical space is free of polymer. In some embodiments, at least some ofthe crystal particles in said three dimensional physical space definingsaid at least one pharmaceutical agent layer are in contact with polymerparticles present in a polymer layer adjacent to said at least onepharmaceutical agent layer defined by said three-dimensional space freeof polymer.

In some embodiments, the plurality of layers comprises a first polymerlayer comprising a first bioabsorbable polymer and a second polymerlayer comprising a second bioabsorbable polymer, wherein said at leastone layer comprising said pharmaceutical agent is between said firstpolymer layer and said second polymer layer. In some embodiments, firstand second bioabsorbable polymers are the same polymer. In someembodiments, the first and second bioabsorbable polymers are different.In some embodiments, the second polymer layer has at least one contactpoint with at least one particle of said pharmaceutical agent in saidpharmaceutical agent layer and said second polymer layer has at leastone contact point with said first polymer layer.

In some embodiments, the stent has a stent longitudinal axis; and saidsecond polymer layer has a second polymer layer portion along said stentlongitudinal wherein said second layer portion is free of contact withparticles of said pharmaceutical agent. In some embodiments, the devicehas at least one pharmaceutical agent layer defined by athree-dimensional physical space occupied by crystal particles of saidpharmaceutical agent and said three dimensional physical space is freeof polymer.

The second polymer layer may have a layer portion defined along alongitudinal axis of the stent, said polymer layer portion having athickness less than said maximum thickness of said second polymer layer;wherein said portion is free of contact with particles of saidpharmaceutical agent.

The polymer layer portion may be a sub layer which, at least in part,extends along the abluminal surface of the stent along the longitudinalaxis of the stent (where the longitudinal axis of the stent is thecentral axis of the stent along its tubular length). For example, when acoating is removed from the abluminal surface of the stent, such as whenthe stent is cut along its length, flattened, and the coating is removedby scraping the coating off using a scalpel, knife or other sharp tool,the coating that is removed (despite having a pattern consistent withthe stent pattern) has a layer that can be shown to have thecharacteristics described herein. This may be shown by sampling multiplelocations of the coating that is representative of the entire coating.

Alternatively, and/or additionally, since stents are generally comprisedof a series of struts and voids. The methods provided hereinadvantageously allow for coatings extending around each strut, thelayers of coating are likewise disposed around each strut. Thus, apolymer layer portion may be a layer which, at least, extends aroundeach strut a distance from said strut (although the distance may varywhere the coating thickness on the abluminal surface is different thanthe coating thickness on the luminal and/or sidewalls).

In some embodiments, the stent comprises at least one strut having astrut length along said stent longitudinal axis, wherein said secondlayer portion extends substantially along said strut length. In someembodiments, the stent has a stent length along said stent longitudinalaxis and said second layer portion extends substantially along saidstent length.

In some embodiments, the stent comprises at least five struts, eachstrut having a strut length along said stent longitudinal axis, whereinsaid second layer portion extends substantially along substantially thestrut length of at least two struts. In some embodiments, the stentcomprises at least five struts, each strut having a strut length alongsaid stent longitudinal axis, wherein said second layer portion extendssubstantially along substantially the strut length of at least threestruts. In some embodiments, the stent comprises at least five struts,each strut having a strut length along said stent longitudinal axis,wherein said second layer portion extends substantially alongsubstantially the strut length of least four struts. In someembodiments, the stent comprises at least five struts, each strut havinga strut length along said stent longitudinal axis, wherein said secondlayer portion extends substantially along substantially the strut lengthof all said at least five struts. In some embodiments, the stent has astent length along said stent longitudinal axis and said second layerportion extends substantially along said stent length.

In some embodiments, the stent has a stent length along said stentlongitudinal axis and said second layer portion extends along at least50% of said stent length. In some embodiments, the stent has a stentlength along said stent longitudinal axis and said second layer portionextends along at least 75% of said stent length. In some embodiments,the stent has a stent length along said stent longitudinal axis and saidsecond layer portion extends along at least 85% of said stent length. Insome embodiments, the stent has a stent length along said stentlongitudinal axis and said second layer portion extends along at least90% of said stent length. In some embodiments, the stent has a stentlength along said stent longitudinal axis and said second layer portionextends along at least 99% of said stent length.

In some embodiments, the laminate coating has a total thickness and saidsecond polymer layer portion has a thickness of from about 0.01% toabout 10% of the total thickness of said laminate coating. In someembodiments, the laminate coating has a total thickness and saidhorizontal second polymer layer portion has a thickness of from about 1%to about 5% of the total thickness of said laminate coating. In someembodiments, the laminate coating has a total thickness of from about 5μm to about 50 μm and said horizontal second polymer layer portion has athickness of from about 0.001 μm to about 5 μm. In some embodiments, thelaminate coating has a total thickness of from about 10 μm to about 20μm and said second polymer layer portion has a thickness of from about0.01 μm to about 5 μm.

In some embodiments, the laminate coating is at least 25% by volumepharmaceutical agent. In some embodiments, the laminate coating is atleast 35% by volume pharmaceutical agent. In some embodiments, thelaminate coating is about 50% by volume pharmaceutical agent.

In some embodiments, at least a portion of the pharmaceutical agent ispresent in a phase separate from one or more phases formed by saidpolymer.

In some embodiments, the pharmaceutical agent is at least 50%crystalline. In some embodiments, the pharmaceutical agent is at least75% crystalline. In some embodiments, the pharmaceutical agent is atleast 90% crystalline. In some embodiments, the pharmaceutical agent isat least 95% crystalline. In some embodiments, the pharmaceutical agentis at least 99% crystalline.

In some embodiments, the stent has a stent longitudinal length and thecoating has a coating outer surface along said stent longitudinallength, wherein said coating comprises pharmaceutical agent incrystalline form present in the coating below said coating outersurface. In some embodiments, the stent has a stent longitudinal lengthand the coating has a coating outer surface along said stentlongitudinal length, wherein said coating comprises pharmaceutical agentin crystalline form present in the coating up to at least 1 μm belowsaid coating outer surface. In some embodiments, the stent has a stentlongitudinal length and the coating has a coating outer surface alongsaid stent longitudinal length, wherein said coating comprisespharmaceutical agent in crystalline form present in the coating up to atleast 5 μm below said coating outer surface.

In some embodiments, the coating exhibits an X-ray spectrum showing thepresence of said pharmaceutical agent in crystalline form. In someembodiments, the coating exhibits a Raman spectrum showing the presenceof said pharmaceutical agent in crystalline form. In some embodiments,the coating exhibits a Differential Scanning Calorimetry (DSC) curveshowing the presence of said pharmaceutical agent in crystalline form.The device of Claims 36-38, wherein said coating exhibits Wide AngleX-ray Scattering (WAXS) spectrum showing the presence of saidpharmaceutical agent in crystalline form. In some embodiments, thecoating exhibits a wide angle radiation scattering spectrum showing thepresence of said pharmaceutical agent in crystalline form. In someembodiments, the coating exhibits an Infra Red (IR) spectrum showing thepresence of said pharmaceutical agent in crystalline form.

In some embodiments, the stent has a stent longitudinal axis and a stentlength along said stent longitudinal axis, wherein said coating isconformal to the stent along substantially said stent length.

In some embodiments, the stent has a stent longitudinal axis and a stentlength along said stent longitudinal axis, wherein said coating isconformal to the stent along at least 75% of said stent length. In someembodiments, the stent has a stent longitudinal axis and a stent lengthalong said stent longitudinal axis, wherein said coating is conformal tothe stent along at least 85% of said stent length. In some embodiments,the stent has a stent longitudinal axis and a stent length along saidstent longitudinal axis, wherein said coating is conformal to the stentalong at least 90% of said stent length. In some embodiments, the stenthas a stent longitudinal axis and a stent length along said stentlongitudinal axis, wherein said coating is conformal to the stent alongat least 95% of said stent length. In some embodiments, the stent has astent longitudinal axis and a stent length along said stent longitudinalaxis, wherein said coating is conformal to the stent along at least 99%of said stent length.

In some embodiments, the stent has a stent longitudinal axis and aplurality of struts along said stent longitudinal axis, wherein saidcoating is conformal to at least 50% of said struts. In someembodiments, the stent has a stent longitudinal axis and a plurality ofstruts along said stent longitudinal axis, wherein said coating isconformal to at least 75% of said struts. In some embodiments, the stenthas a stent longitudinal axis and a plurality of struts along said stentlongitudinal axis, wherein said coating is conformal to at least 90% ofsaid struts. In some embodiments, the stent has a stent longitudinalaxis and a plurality of struts along said stent longitudinal axis,wherein said coating is conformal to at least 99% of said struts. Insome embodiments, the stent has a stent longitudinal axis and a stentlength along said stent longitudinal axis, wherein an electronmicroscopy examination of the device shows said coating is conformal tosaid stent along at least 90% of said stent length.

In some embodiments, the stent has a stent longitudinal axis and a stentlength along said stent longitudinal axis, wherein said coating has asubstantially uniform thickness along substantially said stent length.

In some embodiments, the stent has a stent longitudinal axis and a stentlength along said stent longitudinal axis, wherein said coating has asubstantially uniform thickness along at least 75% of said stent length.In some embodiments, the stent has a stent longitudinal axis and a stentlength along said stent longitudinal axis, wherein said coating has asubstantially uniform thickness along at least 95% of said stent length.

In some embodiments, the stent has a stent longitudinal axis and a stentlength along said stent longitudinal axis, wherein said coating has anaverage thickness determined by an average calculated from coatingthickness values measured at a plurality of points along said stentlongitudinal axis; wherein a thickness of the coating measured at anypoint along stent longitudinal axis is from about 75% to about 125% ofsaid average thickness. In some embodiments, the stent has a stentlongitudinal axis and a stent length along said stent longitudinal axis,wherein said coating has an average thickness determined by an averagecalculated from coating thickness values measured at a plurality ofpoints along said stent longitudinal axis; wherein a thickness of thecoating measured at any point along stent longitudinal axis is fromabout 95% to about 105% of said average thickness.

Provided herein is a device comprising: a stent; and a plurality oflayers that form a laminate coating on said stent, wherein a first layercomprises a first bioabsorbable polymer, a second layer comprises apharmaceutical agent, a third layer comprises a second bioabsorbablepolymer, a fourth layer comprises the pharmaceutical agent, and a fifthlayer comprises a third bioabsorbable polymer, wherein thepharmaceutical agent is selected from rapamycin, a prodrug, aderivative, an analog, a hydrate, an ester, and a salt thereof, andwherein at least a portion of the pharmaceutical agent is in crystallineform.

In some embodiments, at least two of said first bioabsorbable polymer,said second bioabsorbable polymer and said third bioabsorbable polymerare the same polymer. In some embodiments, the first bioabsorbablepolymer, the second bioabsorbable polymer and the third bioabsorbablepolymer are the same polymer. In some embodiments, at least two of saidfirst bioabsorbable polymer, said second bioabsorbable polymer and saidthird bioabsorbable polymer are different polymers. In some embodiments,the first bioabsorbable polymer, said second bioabsorbable polymer andsaid third bioabsorbable polymer are different polymers.

In some embodiments, the third layer has at least one contact point withparticles of said pharmaceutical agent in said second layer; and saidthird layer has at least one contact point with said first layer.

In some embodiments, at least two of the first polymer, the secondpolymer, and the third polymer are the same polymer, and wherein saidsame polymer comprises a PLGA copolymer. In some embodiments, the thirdpolymer has an in vitro dissolution rate higher than the in vitrodissolution rate of the first polymer. In some embodiments, the thirdpolymer is PLGA copolymer with a ratio of about 40:60 to about 60:40 andthe first polymer is a PLGA copolymer with a ratio of about 70:30 toabout 90:10. In some embodiments, the third polymer is PLGA copolymerhaving a molecular weight of about 10 kD and the second polymer is aPLGA copolymer having a molecular weight of about 19 kD.

In some embodiments, measuring the in vitro dissolution rate of saidpolymers comprises contacting the device with elution media anddetermining polymer weight loss at one or more selected time points. Insome embodiments, measuring the in vitro dissolution rate of saidpolymers comprises contacting the device with elution media anddetermining polymer weight loss at one or more selected time points.

Provided herein is a device, comprising: a stent; and a coating on saidstent comprising a first bioabsorbable polymer, a second bioabsorbablepolymer; and pharmaceutical agent selected from rapamycin, a prodrug, aderivative, an analog, a hydrate, an ester, and a salt thereof whereinat least a portion of the pharmaceutical agent is in crystalline form,and wherein the first polymer has an in vitro dissolution rate higherthan the in vitro dissolution rate of the second polymer.

In some embodiments, the first polymer is PLGA copolymer with a ratio ofabout 40:60 to about 60:40 and the second polymer is a PLGA copolymerwith a ratio of about 70:30 to about 90:10. In some embodiments, thefirst polymer is PLGA copolymer having a molecular weight of about 10 kDand the second polymer is a PLGA copolymer having a molecular weight ofabout 19 kD. In some embodiments, measuring the in vitro dissolutionrate of said polymers comprises contacting the device with elution mediaand determining polymer weight loss at one or more selected time points.

Provided herein is a device comprising a stent; and a plurality oflayers that form a laminate coating on said stent; wherein at least oneof said layers comprises a first bioabsorbable polymer, at least one ofsaid layers comprises a second bioabsorbable polymer, and at least oneof said layers comprises one or more active agents; wherein at least aportion of the active agent is in crystalline form, and wherein thefirst polymer has an in vitro dissolution rate higher than the in vitrodissolution rate of the second polymer.

Provided herein is a device comprising a stent; and a plurality oflayers that form a laminate coating on said stent; wherein at least oneof said layers comprises a first bioabsorbable polymer, at least one ofsaid layers comprises a second bioabsorbable polymer, and at least oneof said layers comprises a pharmaceutical agent selected from rapamycin,a prodrug, a derivative, an analog, a hydrate, an ester, and a saltthereof, wherein at least a portion of the pharmaceutical agent is incrystalline form and wherein the first polymer has an in vitrodissolution rate higher than the in vitro dissolution rate of the secondpolymer.

In some embodiments, the first polymer is PLGA copolymer with a ratio ofabout 40:60 to about 60:40 and the second polymer is a PLGA copolymerwith a ratio of about 70:30 to about 90:10. In some embodiments, thefirst polymer is PLGA copolymer having a molecular weight of about 10 kDand the second polymer is a PLGA copolymer having a molecular weight ofabout 19 kD. In some embodiments, measuring the in vitro dissolutionrate comprises contacting the device with elution media and determiningpolymer weight loss at one or more selected time points.

Provided herein is a device comprising a stent; and a plurality oflayers that form a laminate coating on said stent; wherein at least oneof said layers comprises a bioabsorbable polymer, at least one of saidlayers comprises a first active agent and at least one of said layerscomprises a second active agent; wherein at least a portion of firstand/or second active agents is in crystalline form.

In some embodiments, the bioabsorbable polymer is selected from thegroup PLGA, PGA poly(glycolide), LPLA poly(1-lactide), DLPLApoly(dl-lactide), PCL poly(e-caprolactone) PDO, poly(dioxolane) PGA-TMC,85/15 DLPLG p(dl-lactide-co-glycolide), 75/25 DLPL, 65/35 DLPLG, 50/50DLPLG, TMC poly(trimethylcarbonate), p(CPP:SA)poly(1,3-bis-p-(carboxyphenoxy)propane-co-sebacic acid). In someembodiments, the polymer comprises an intimate mixture of two or morepolymers.

In some embodiments, the first and second active agents areindependently selected from pharmaceutical agents and active biologicalagents.

In some embodiments, the stent is formed of stainless steel material. Insome embodiments, the stent is formed of a material comprising a cobaltchromium alloy. In some embodiments, the stent is formed from a materialcomprising the following percentages by weight: about 0.05 to about 0.15C, about 1.00 to about 2.00 Mn, about 0.04 Si, about 0.03 P, about 0.3S, about 19.0 to about 21.0 Cr, about 9.0 to about 11.0 Ni, about 14.0to about 16.00 W, about 3.0 Fe, and Bal. Co. In some embodiments, thestent is formed from a material comprising at most the followingpercentages by weight: about 0.025 C, about 0.15 Mn, aboout 0.15 Si,about 0.015 P, about 0.01 S, about 19.0 to about 21.0 Cr, about 33 toabout 37 Ni, about 9.0 to about 10.5 Mo, about 1.0 Fe, about 1.0 Ti, andBal. Co. In some embodiments, the stent is formed from a materialcomprising L605 alloy.

In some embodiments, the stent has a thickness of from about 50% toabout 90% of a total thickness of said device. In some embodiments, thedevice has a thickness of from about 20 μm to about 500 μm. In someembodiments, the device has a thickness of about 90 μm or less. In someembodiments, the laminate coating has a thickness of from about 5 μm toabout 50 μm. In some embodiments, the laminate coating has a thicknessof from about 10 μm to about 20 μm. In some embodiments, the stent has athickness of from about 50 μm to about 80 μm.

Provided herein is a device comprising: a stent, wherein the stent isformed from a material comprising the following percentages by weight:0.05-0.15 C, 1.00-2.00 Mn, 0.040 Si, 0.030 P, 0.3 S, 19.00-21.00 Cr,9.00-11.00 Ni, 14.00-16.00 W, 3.00 Fe, and Bal. Co; and a plurality oflayers that form a laminate coating on said stent, wherein a first layercomprises a first bioabsorbable polymer, a second layer comprises apharmaceutical agent, a third layer comprises a second bioabsorbablepolymer, a fourth layer comprises the pharmaceutical agent, and a fifthlayer comprises a third bioabsorbable polymer, wherein thepharmaceutical agent is selected from rapamycin, a prodrug, aderivative, an analog, a hydrate, an ester, and a salt thereof, whereinat least a portion of the pharmaceutical agent is in crystalline form,and wherein at least one of said first polymer, second polymer and thirdpolymer comprises a PLGA copolymer.

In some embodiments, the device has a pharmaceutical agent content offrom about 0.5 μg/mm to about 20 μg/mm. In some embodiments, the devicehas a pharmaceutical agent content of from about 8 μg/mm to about 12μg/mm. In some embodiments, the device has a pharmaceutical agentcontent of from about 5 μg to about 500 μg. In some embodiments, thedevice has a pharmaceutical agent content of from about 100 μg to about160 μg. In some embodiments, the device has a pharmaceutical agentcontent of from about 100 μg to about 160 μg.

Content is expressed herein in units of μg/mm, however, this may simplybe converted to μg/mm² or another amount per area (e.g., μg/cm²).

Provided herein is a method of preparing a device comprising a stent anda plurality of layers that form a laminate coating on said stent; saidmethod comprising: (a) providing a stent; (b) forming a plurality oflayers on said stent to form said laminate coating on said stent;wherein at least one of said layers comprises a bioabsorbable polymerand at least one of said layers comprises one or more active agents;wherein at least a portion of the active agent is in crystalline form.

Provided herein is a method of preparing a device comprising a stent anda plurality of layers that form a laminate coating on said stent; saidmethod comprising: (a) providing a stent; (b) forming a plurality oflayers to form said laminate coating on said stent; wherein at least oneof said layers comprises a bioabsorbable polymer and at least one ofsaid layers comprises a pharmaceutical agent selected from rapamycin, aprodrug, a derivative, an analog, a hydrate, an ester, and a saltthereof, wherein at least a portion of the pharmaceutical agent is incrystalline form.

Provided herein is a method of preparing a device comprising a stent anda plurality of layers that form a laminate coating on said stent; saidmethod comprising: (a) providing a stent; (b) forming a plurality oflayers to form said laminate coating on said stent; wherein at least oneof said layers comprises a bioabsorbable polymer and at least one ofsaid layers comprises a pharmaceutical agent selected from rapamycin, aprodrug, a derivative, an analog, a hydrate, an ester, and a saltthereof, wherein at least a portion of the pharmaceutical agent is incrystalline form, wherein said method comprises forming at least onepharmaceutical agent layer defined by a three-dimensional physical spaceoccupied by crystal particles of said pharmaceutical agent and saidthree dimensional physical space is free of polymer.

Provided herein is a method of preparing a device comprising a stent anda plurality of layers that form a laminate coating on said stent; saidmethod comprising: (a) providing a stent; (b) discharging at least onepharmaceutical agent and/or at least one active biological agent in drypowder form through a first orifice; (c) forming a supercritical or nearsupercritical fluid solution comprising at least one supercritical fluidsolvent and at least one polymer and discharging said supercritical ornear supercritical fluid solution through a second orifice underconditions sufficient to form solid particles of the polymer; (d)depositing the polymer and pharmaceutical agent and/or active biologicalagent particles onto said substrate, wherein an electrical potential ismaintained between the substrate and the polymer and pharmaceuticalagent and/or active biological agent particles, thereby forming saidcoating; and (e) sintering said polymer under conditions that do notsubstantially modify a morphology of said pharmaceutical agent and/oractivity of said biological agent.

In some embodiments, step (b) comprises discharging a pharmaceuticalagent selected from rapamycin, a prodrug, a derivative, an analog, ahydrate, an ester, and a salt thereof, wherein at least a portion of thepharmaceutical agent is in crystalline form. In some embodiments, step(c) comprises forming solid particles of a bioabsorbable polymer.

In some embodiments, step (e) comprises forming a polymer layer having alength along a horizontal axis of said device wherein said polymer layerhas a layer portion along said length, wherein said layer portion isfree of pharmaceutical agent.

In some embodiments, step (e) comprises contacting said polymer with adensified fluid. In some embodiments, step (e) comprises contacting saidpolymer with a densified fluid for a period of time at a temperature offrom about 5° C. and 150° C. and a pressure of from about 10 psi toabout 500 psi. In some embodiments, step (e) comprises contacting saidpolymer with a densified fluid for a period of time at a temperature offrom about 25° C. and 95° C. and a pressure of from about 25 psi toabout 100 psi. In some embodiments, step (e) comprises contacting saidpolymer with a densified fluid for a period of time at a temperature offrom about 50° C. and 85° C. and a pressure of from about 35 psi toabout 65 psi.

Provided herein is a method of preparing a device comprising a stent anda plurality of layers that form a laminate coating on said stent; saidmethod comprising: (a) providing a stent; (b) forming a supercritical ornear supercritical fluid solution comprising at least one supercriticalfluid solvent and a first polymer, discharging said supercritical ornear supercritical fluid solution under conditions sufficient to formsolid particles of said first polymer, depositing said first polymerparticles onto said stent, wherein an electrical potential is maintainedbetween the stent and the first polymer, and sintering said firstpolymer; (c) depositing pharmaceutical agent particles in dry powderform onto said stent, wherein an electrical potential is maintainedbetween the stent and said pharmaceutical agent particles; and (d)forming a supercritical or near supercritical fluid solution comprisingat least one supercritical fluid solvent and a second polymer anddischarging said supercritical or near supercritical fluid solutionunder conditions sufficient to form solid particles of said secondpolymer, wherein an electrical potential is maintained between the stentand the second polymer, and sintering said second polymer.

In some embodiments, step (c) and step (d) are repeated at least once.In some embodiments, steps (c) and step (d) are repeated 2 to 20 times.

In some embodiments, the pharmaceutical agent is selected fromrapamycin, a prodrug, a derivative, an analog, a hydrate, an ester, anda salt thereof, wherein at least a portion of the pharmaceutical agentis in crystalline form. In some embodiments, the first and secondpolymers are bioabsorbable.

In some embodiments, step (d) comprises forming a polymer layer having alength along a horizontal axis of said device wherein said polymer layerhas a layer portion along said length, wherein said layer portion isfree of pharmaceutical agent.

In some embodiments, sintering said first and/or sintering said secondpolymer comprises contacting said first and/or second polymer with adensified fluid.

In some embodiments, the contacting step is carried out for a period offrom about 1 minute to about 60 minutes. In some embodiments, thecontacting step is carried out for a period of from about 10 minutes toabout 30 minutes.

In some embodiments, maintaining said electrical potential between saidpolymer particles and or pharmaceutical agent particles and said stentcomprises maintaining a voltage of from about 5 kvolts to about 100kvolts. In some embodiments, maintaining said electrical potentialbetween said polymer particles and or pharmaceutical agent particles andsaid stent comprises maintaining a voltage of from about 20 kvolts toabout 30 kvolts.

Provided herein is a device prepared by a process comprising a method asdescribed herein.

Provided herein is method of treating a subject comprising delivering adevice as described herein in a body lumen of the subject.

Provided herein is a method of treating a subject comprising deliveringin the body of the subject a device comprising: a stent, wherein thestent is formed from a material comprising the following percentages byweight: 0.05-0.15 C, 1.00-2.00 Mn, 0.040 Si, 0.030 P, 0.3 S, 19.00-21.00Cr, 9.00-11.00 Ni, 14.00-16.00 W, 3.00 Fe, and Bal. Co; and a pluralityof layers that form a laminate coating on said stent, wherein a firstlayer comprises a first bioabsorbable polymer, a second layer comprisesa pharmaceutical agent, a third layer comprises a second bioabsorbablepolymer, a fourth layer comprises the pharmaceutical agent, and a fifthlayer comprises a third bioabsorbable polymer, wherein thepharmaceutical agent is selected from rapamycin, a prodrug, aderivative, an analog, a hydrate, an ester, and a salt thereof, whereinat least a portion of the pharmaceutical agent is in crystalline form,and wherein at least one of said first polymer, second polymer and thirdpolymer comprises a PLGA copolymer.

In some embodiments, the device has a pharmaceutical agent content offrom about 0.5 μg/mm to about 20 μg/mm. In some embodiments, the devicehas a pharmaceutical agent content of from about 8 μg/mm to about 12μg/mm. In some embodiments, the device has a pharmaceutical agentcontent of from about 100 μg to about 160 μg. In some embodiments, thedevice has a pharmaceutical agent content of from about 120 μg to about150 μg.

In some embodiments, the device has an initial pharmaceutical agentamount and the amount of pharmaceutical agent delivered by said deviceto vessel wall tissue of said subject is higher than the amount ofpharmaceutical agent delivered by a conventional drug eluting stenthaving the same initial pharmaceutical agent content as the initialpharmaceutical agent content of said device. In some embodiments, theamount of pharmaceutical agent delivered by said device to vessel walltissue of said subject is at least 25% more that the amount ofpharmaceutical agent delivered to vessel wall tissue of said subject bysaid conventional drug eluting stent. In some embodiments, the methodcomprises treating restenosis in a blood vessel of said the subject. Insome embodiments, the subject is selected from a pig, a rabbit and ahuman.

“Vessel wall tissue” as used herein is shown in FIG. 11, which depictsthe tissue surrounding the lumen of a vessel, including the endothelium,neointima, tunica media, IEL (internal elastic lamina), EEL (externalelastic lamina), and the tunica adventitia.

Provided herein is a device comprising: a stent; and a plurality oflayers on said stent; wherein at least one of said layers comprises abioabsorbable polymer and at least one of said layers comprises apharmaceutical agent selected from rapamycin, a prodrug, a derivative,an analog, a hydrate, an ester, and a salt thereof, wherein said deviceprovides an in vitro pharmaceutical agent elution profile wherein saidelution profile shows about 5% to about 25% of pharmaceutical agent iseluted one day after the device is contacted with elution media; 15% toabout 45% of pharmaceutical agent is eluted 7 days after the device iscontacted with elution media; about 25% to about 60% of pharmaceuticalagent is eluted 14 days after the device is contacted with elutionmedia; about 35% to about 70% of pharmaceutical agent is eluted 21 daysafter the device is contacted with elution media; and about 40% to about100% of pharmaceutical agent is eluted 28 days after the device iscontacted with elution media.

Provided herein is a device comprising a stent; and a plurality oflayers on said stent; wherein at least one of said layers comprises abioabsorbable polymer and at least one of said layers comprises apharmaceutical agent selected from rapamycin, a prodrug, a derivative,an analog, a hydrate, an ester, and a salt thereof, wherein said deviceprovides an in vitro pharmaceutical agent elution profile wherein saidelution profile shows about 7% to about 15% of pharmaceutical agent iseluted one day after the device is contacted with elution media; 25% toabout 35% of pharmaceutical agent is eluted 7 days after the device iscontacted with elution media; about 35% to about 55% of pharmaceuticalagent is eluted 14 days after the device is contacted with elutionmedia; about 45% to about 60% of pharmaceutical agent is eluted 21 daysafter the device is contacted with elution media; and about 50% to about70% of pharmaceutical agent is eluted 28 days after the device iscontacted with elution media.

Provided herein is a device comprising a stent; and a plurality oflayers on said stent; wherein at least one of said layers comprises abioabsorbable polymer and at least one of said layers comprises apharmaceutical agent selected from rapamycin, a prodrug, a derivative,an analog, a hydrate, an ester, and a salt thereof, wherein said deviceprovides an in vitro pharmaceutical agent elution profile wherein saidelution profile shows at least 5% of pharmaceutical agent is eluted oneday after the device is contacted with elution media; at least 15% ofpharmaceutical agent is eluted 7 days after the device is contacted withelution media; at least 25% of pharmaceutical agent is eluted 14 daysafter the device is contacted with elution media; at least 30% ofpharmaceutical agent is eluted 21 days after the device is contactedwith elution media; at least 40% of pharmaceutical agent is eluted 28days after the device is contacted with elution media.

Provided herein is a device comprising a stent; and a plurality oflayers on said stent; wherein at least one of said layers comprises abioabsorbable polymer and at least one of said layers comprises apharmaceutical agent selected from rapamycin, a prodrug, a derivative,an analog, a hydrate, an ester, and a salt thereof, wherein said deviceprovides an in vitro pharmaceutical agent elution profile wherein saidelution profile shows about 10% of pharmaceutical agent is eluted oneday after the device is contacted with elution media; about 30% ofpharmaceutical agent is eluted 7 days after the device is contacted withelution media; about 45% of pharmaceutical agent is eluted 14 days afterthe device is contacted with elution media; about 50% of pharmaceuticalagent is eluted 21 days after the device is contacted with elutionmedia; about 60% of pharmaceutical agent is eluted 28 days after thedevice is contacted with elution media.

Provided herein is a device comprising a stent; and a plurality oflayers on said stent; wherein at least one of said layers comprises abioabsorbable polymer and at least one of said layers comprises apharmaceutical agent selected from rapamycin, a prodrug, a derivative,an analog, a hydrate, an ester, and a salt thereof, wherein said deviceprovides an in vitro pharmaceutical agent elution profile wherein saidelution profile shows about 10% to about 75% of pharmaceutical agent iseluted at week 1 after the device is contacted with elution media, about25% to about 85% of pharmaceutical agent is eluted at week 2 and about50% to about 100% of pharmaceutical agent is eluted at week 10.

Provided herein is a device comprising: a stent; and a plurality oflayers on said stent; wherein at least one of said layers comprises abioabsorbable polymer and at least one of said layers comprises apharmaceutical agent selected from rapamycin, a prodrug, a derivative,an analog, a hydrate, an ester, and a salt thereof, wherein said deviceprovides an in vitro pharmaceutical agent elution profile shown in FIG.5.

In some embodiments, the in vitro pharmaceutical agent elution profileis determined by a procedure comprising: (i) contacting the device withan elution media comprising 5% ethanol by volume wherein the pH of themedia is about 7.4 and wherein the device is contacted with the elutionmedia at a temperature of about 37° C.; (ii) optionally agitating theelution media during the contacting step in (i); (iii) removing theelution media at designated time points; and (iv) assaying the removedelution media to determine pharmaceutical agent content.

In some embodiments, the in vitro pharmaceutical agent elution profileis determined by a procedure comprising: (i) contacting the device withan elution media comprising 5% ethanol by volume, wherein the pH of themedia is about 7.4 and wherein the device is contacted with the elutionmedia at a temperature of about 37° C.; (ii) optionally agitating theelution media during the contacting step in (i); (iii) removing saiddevice from the elution media at designated time points; and (iv)assaying the elution media to determine pharmaceutical agent content.

In some embodiments, the in vitro pharmaceutical agent elution profileis determined in the absence of agitation.

In some embodiments, the procedure further comprises: (v) determiningpolymer weight loss by comparing the weight of the device before andafter the contacting step and adjusting for the amount of pharmaceuticalagent eluted into the elution media as determined in step (iv). In someembodiments, step (v) shows at least 50% of polymer is released into themedia after the device is contacted with the media for 90 days or more.In some embodiments, step (v) shows at least 75% of polymer is releasedinto the media after the device is contacted with the media for 90 daysor more.

In some embodiments, step (v) shows at least 85% of polymer is releasedinto the media after the device is contacted with the media for 90 daysor more. In some embodiments, step (v) shows at least 50% of polymer isreleased into the media after the device is contacted with the media forabout 90 days. In some embodiments, step (v) shows at least 75% ofpolymer is released into the media after the device is contacted withthe media for about 90 days. In some embodiments, step (v) shows atleast 85% of polymer is released into the media after the device iscontacted with the media for about 90 days. In some embodiments, step(v) shows at least 95% of polymer is released into the media after thedevice is contacted with the media for about 90 days. In someembodiments, step (v) shows up to 100% of polymer is released into themedia after the device is contacted with the media for about 90 days.

Provided herein is a device comprising: a stent; and a plurality oflayers on said stent; wherein at least one of said layers comprises abioabsorbable polymer and at least one of said layers comprises apharmaceutical agent selected from rapamycin, a prodrug, a derivative,an analog, a hydrate, an ester, and a salt thereof, wherein said deviceprovides an in vitro pharmaceutical agent elution profile wherein saidelution profile shows about 1% to about 35% of pharmaceutical agent iseluted one hour after the device is contacted with elution media; 5% toabout 45% of pharmaceutical agent is eluted 3 hours after the device iscontacted with elution media; about 30% to about 70% of pharmaceuticalagent is eluted 1 day after the device is contacted with elution media;about 40% to about 80% of pharmaceutical agent is eluted 3 days afterthe device is contacted with elution media; about 50% to about 90% ofpharmaceutical agent is eluted 10 days after the device is contactedwith elution media about 55% to about 95% of pharmaceutical agent iseluted 15 days after the device is contacted with elution media; andabout 60% to about 100% of pharmaceutical agent is eluted 20 days afterthe device is contacted with elution media.

Provided herein is a device comprising: a stent; and a plurality oflayers on said stent; wherein at least one of said layers comprises abioabsorbable polymer and at least one of said layers comprises apharmaceutical agent selected from rapamycin, a prodrug, a derivative,an analog, a hydrate, an ester, and a salt thereof, wherein said deviceprovides an in vitro pharmaceutical agent elution profile wherein saidelution profile shows about 5% to about 25% of pharmaceutical agent iseluted one hour after the device is contacted with elution media; 5% toabout 35% of pharmaceutical agent is eluted 3 hours after the device iscontacted with elution media; about 30% to about 65% of pharmaceuticalagent is eluted 1 day after the device is contacted with elution media;about 45% to about 70% of pharmaceutical agent is eluted 3 days afterthe device is contacted with elution media; about 55% to about 85% ofpharmaceutical agent is eluted 10 days after the device is contactedwith elution media about 65% to about 85% of pharmaceutical agent iseluted 15 days after the device is contacted with elution media; andabout 75% to about 100% of pharmaceutical agent is eluted 20 days afterthe device is contacted with elution media.

Provided herein is a device comprising: a stent; and a plurality oflayers on said stent; wherein at least one of said layers comprises abioabsorbable polymer and at least one of said layers comprises apharmaceutical agent selected from rapamycin, a prodrug, a derivative,an analog, a hydrate, an ester, and a salt thereof, wherein said deviceprovides an in vitro pharmaceutical agent elution profile shown in FIG.9.

In some embodiments, the in vitro pharmaceutical agent elution profileis determined by a procedure comprising: (i) contacting the device withan elution media comprising ethanol and phosphate buffered salinewherein the pH of the media is about 7.4 and wherein the device iscontacted with the elution media at a temperature of about 37° C.; (ii)optionally agitating the elution media during the contacting step in(i); (iii) removing the elution media at designated time points; and(iv) assaying the removed elution media to determine pharmaceuticalagent content.

In some embodiments, the in vitro pharmaceutical agent elution profileis determined by a procedure comprising: (i) contacting the device withan elution media comprising ethanol and phosphate buffered salinewherein the pH of the media is about 7.4 and wherein the device iscontacted with the elution media at a temperature of about 37° C.; (ii)optionally agitating the elution media during the contacting step in(i); (iii) removing said device from the elution media at designatedtime points; and (iv) assaying the elution media to determinepharmaceutical agent content.

In some embodiments, the in vitro pharmaceutical agent elution profileis determined in the absence of agitation.

In some embodiments, the procedure further comprises: (v) determiningpolymer weight loss by comparing the weight of the device before andafter the contacting step and adjusting for the amount of pharmaceuticalagent eluted into the elution media as determined in step iv. The deviceof claim 160 wherein step v shows at least 50% of polymer is releasedinto the media after the device is contacted with the media for 90 daysor more.

In some embodiments, step (v) shows at least 75% of polymer is releasedinto the media after the device is contacted with the media for 90 daysor more. In some embodiments, step (v) shows at least 85% of polymer isreleased into the media after the device is contacted with the media for90 days or more. In some embodiments, step (v) shows at least 50% ofpolymer is released into the media after the device is contacted withthe media for about 90 days. In some embodiments, step (v) shows atleast 75% of polymer is released into the media after the device iscontacted with the media for about 90 days. In some embodiments, step(v) shows at least 85% of polymer is released into the media after thedevice is contacted with the media for about 90 days. In someembodiments, step (v) shows at least 95% of polymer is released into themedia after the device is contacted with the media for about 90 days.

Provided herein is a device comprising: a stent; and a coatingcomprising a pharmaceutical agent selected from rapamycin, a prodrug, aderivative, ester and a salt thereof and a polymer wherein the coatinghas an initial pharmaceutical agent amount; wherein when said device isdelivered in a body lumen of a subject the pharmaceutical agent isdelivered in vessel wall tissue of the subject as follows: from about0.1% to about 35% of the initial pharmaceutical agent amount isdelivered in the subject's vessel wall tissue one week after the deviceis delivered in the subject's body; and from about 0.5% to about 50% ofthe initial pharmaceutical agent amount is delivered in the subject'svessel wall tissue two weeks after the device is delivered in thesubject's body.

In some embodiments, the amount delivered to the subject's lumen isobtained by adding pharmaceutical agent present alone in said subject'svessel wall tissue and pharmaceutical agent delivered together with saidpolymer. In some embodiments, the subject is a human.

In some embodiments, subject is a pig and the amount of pharmaceuticalagent delivered in the subject's vessel wall tissue is determined asfollows: delivering the device in the pig's blood vessel lumen;euthanizing the pig at predetermined period of time after the device isdelivered in the pig's blood vessel lumen and explanting the device;measuring the amount of pharmaceutical agent delivered in the vesselwall tissue.

Provided herein, a device comprising: a stent; and a coating comprisinga pharmaceutical agent selected from rapamycin, a prodrug, a derivative,an analog, a hydrate, an ester, and a salt thereof and a bioabsorbablepolymer wherein the coating has an initial pharmaceutical agent contentof about 1 μg/mm to about 15 μg/mm; wherein said device provides an areaunder a curve (AUC) for content of pharmaceutical agent delivered in thevessel wall tissue of a subject over time as follows: from about 0.05(μg/mm)*day to about 1 (μg/mm)*day when AUC is calculated from the timethe device is delivered in a subject's body to one day after the deviceis delivered in the subject's body; from about 5 (μg/mm)*day to about 10(μg/mm)*day when AUC is calculated starting after the first week thedevice is delivered in the subject's body through the second week afterthe device is delivered in the subject's body; from about 10 (μg/mm)*dayto about 20 (μg/mm)*day when AUC is calculated starting after the secondweek the device is delivered in the subject's body through the fourthweek after the device is delivered in the subject's body; and an AUClast of from about 40 (μg/mm)*day to about 60 (μg/mm)*day.

Provided herein is a device comprising: a stent; and a coatingcomprising a pharmaceutical agent selected from rapamycin, a prodrug, aderivative, an analog, a hydrate, an ester, and a salt thereof and abioabsorbable polymer wherein the coating has an initial polymer amount;wherein when said device is delivered in a body lumen of a subject about75% of polymer is released from the device 90 days or more after thedevice is delivered in the body lumen of the subject.

Provided herein is a device comprising: a stent; and a coatingcomprising a pharmaceutical agent selected from rapamycin, a prodrug, aderivative, an analog, a hydrate, an ester, and a salt thereof and abioabsorbable polymer wherein the coating has an initial polymer amount;wherein when said device is delivered in a body lumen of a subject about85% of polymer is released from the device about 90 days after thedevice is delivered in the body lumen of the subject.

Provided herein is a device comprising: a stent; and a coatingcomprising a pharmaceutical agent selected from rapamycin, a prodrug, aderivative, an analog, a hydrate, an ester, and a salt thereof and abioabsorbable polymer wherein the coating has an initial polymer amount;wherein when said device is delivered in a body lumen of a subject atleast about 75% of polymer is released from the device about 90 daysafter the device is delivered in the body lumen of the subject.

Provided herein is a device comprising: a stent; and a coatingcomprising a pharmaceutical agent selected from rapamycin, a prodrug, aderivative, an analog, a hydrate, an ester, and a salt thereof and abioabsorbable polymer wherein the coating has an initial polymer amount;wherein when said device is delivered in a body lumen of a subject about100% of polymer is released from the device about 90 days after thedevice is delivered in the body lumen of the subject.

In some embodiments, the subject is a human. In some embodiments, thesubject is a pig and the amount of polymer released from the device isdetermined as follows: delivering the device in the pig's blood vessellumen; euthanizing the pig at predetermined period of time after thedevice is delivered in the pig's blood vessel lumen and explanting thedevice; and measuring the amount of polymer released from the device.

In some embodiments, measuring the amount of polymer released from thedevice comprises LC/MS/MS measurements. In some embodiments, measuringthe amount released from the device comprises weight loss measurement.In some embodiments, weight loss measurement comprises measuring anamount of polymer remaining in the device and subtracting said remainingamount from the initial amount present in the device prior to deliveringthe device to the pig's blood vessel lumen.

Provided herein is a device comprising a stent; and a plurality oflayers on said stent; wherein at least one of said layers comprises abioabsorbable polymer and at least one of said layers comprises apharmaceutical agent selected from rapamycin, a prodrug, a derivative,an analog, a hydrate, an ester, and a salt thereof, wherein the devicehas an initial pharmaceutical agent content of about 1 μg/mm to about 15μg/mm; wherein when said device is delivered in a body lumen of asubject said device provides a blood concentration within 60 minutesfrom delivery of said device to the subject's body lumen that is fromabout 1% to about 50% of the blood concentration provided by aconventional drug eluting stent delivered to the subject under similarconditions.

Provided herein is a device comprising a stent; and a plurality oflayers on said stent; wherein at least one of said layers comprises abioabsorbable polymer and at least one of said layers comprises apharmaceutical agent selected from rapamycin, a prodrug, a derivative,an analog, a hydrate, an ester, and a salt thereof, wherein the devicehas an initial pharmaceutical agent content of about 1 μg/mm to about 15μg/mm; wherein when said device is delivered in a body lumen of asubject said device provides a blood concentration within 60 minutesfrom delivery of said device to the subject's body lumen that is fromabout 11% to about 20% of the blood concentration provided by aconventional drug eluting stent delivered to the subject under similarconditions.

Provided herein is a device comprising a stent; and coating on saidstent; wherein said coating comprises a bioabsorbable polymer and apharmaceutical agent selected from rapamycin, a prodrug, a derivative,an analog, a hydrate, an ester, and a salt thereof, wherein the devicehas an initial pharmaceutical agent content of about 1 μg/mm to about 15μg/mm; wherein when said device is delivered in a body lumen of asubject said device provides about the same blood concentration over thefirst 72 hours from delivery of said device to the subject's body lumen.

In some embodiments, the blood concentration during the first 72 hoursfrom delivery of said device to the subject's body lumen remains between75% and 125% of an average blood concentration calculated over the first72 hours from delivery of said device to the subject's body lumen. Insome embodiments, the average blood concentration is from about 0.05ng/mL to about 0.5 ng/mL. In some embodiments, the device provides anAUC for blood concentration over a period of 72 hours after the deviceis delivered to the subject's body lumen of from about 2 (ng/mL)*hour toabout 20 (ng/mL)*hour.

In some embodiments, the device provides an AUC for blood concentrationover a period of 72 hours after the device is delivered to the subject'sbody lumen of from about 4 (ng/mL)*hour to about 10 (ng/mL)*hour. Insome embodiments, at least part of pharmaceutical agent is incrystalline form. In some embodiments, the pharmaceutical agent isprovided at a reduced dose compared to a conventional drug elutingstent. In some embodiments, at least one of said layers comprises a PLGAbioabsorbable polymer.

In some embodiments, the pharmaceutical agent in said device has a shelfstability of at least 12 months.

In some embodiments, the device provides an in vitro pharmaceuticalagent elution profile comparable to first order kinetics.

In some embodiments, the device provides pharmaceutical agent tissueconcentration of at least twice the tissue concentration provided by aconventional stent. In some embodiments, the device provides apharmaceutical agent tissue concentration of at least 5 times greaterthan the tissue concentration provided by a conventional stent. In someembodiments, the device provides a pharmaceutical agent tissueconcentration of at least 25 times greater than the tissue concentrationprovided by a conventional stent. In some embodiments, the deviceprovides a pharmaceutical agent tissue concentration of at least 100times greater than the tissue concentration provided by a conventionalstent.

In some embodiments, about 50% of said polymer is resorbed within 45-90days after an angioplasty procedure wherein said device is delivered ina subject's body. In some embodiments, about 75% of said polymer isresorbed within 45-90 days after an angioplasty procedure wherein saiddevice is delivered in a subject's body. In some embodiments, about 95%of said polymer is resorbed within 45-90 days after an angioplastyprocedure wherein said device is delivered in a subject's body.

In some embodiments, 99% of said polymer is resorbed within 45-90 daysafter an angioplasty procedure wherein said device is delivered in asubject's body.

In some embodiments, the device provides reduced inflammation over thecourse of polymer resorbtion compared to a conventional stent.

Provided herein is a method of treating a subject comprising deliveringa device as described herein in a body lumen.

Provided herein, is a method of treating a subject comprising deliveringin the body of the subject a device comprising: a stent; and a coatingcomprising a pharmaceutical agent selected from rapamycin, a prodrug, aderivative, an analog, a hydrate, an ester, and a salt thereof and apolymer wherein the coating has an initial pharmaceutical agent amount;wherein said device is delivered in a body lumen of the subject and thepharmaceutical agent is delivered in vessel wall tissue of the subjectas follows: i. from about 0.05% to about 35% of the initialpharmaceutical agent amount is delivered in the subject's vessel walltissue one week after the device is delivered in the subject's body; andii. from about 0.5% to about 50% of the initial pharmaceutical agentamount is delivered in the subject's vessel wall tissue two weeks afterthe device is delivered in the subject's body.

In some embodiments, the device provides reduced inflammation over thecourse of polymer resorbtion.

In some embodiments, the presence of crystallinity is shown by at leastone of XRD, Raman Spectroscopy, Infrared analytical methods, and DSC.

In some embodiments, the coating on an abluminal surface of said stenthas a greater thickness than coating on a luminal surface of said stent.In some embodiments, the ratio of coating on the abluminal surface tocoating on the luminal surface of the device is 80:20. In someembodiments, the ratio of coating on the abluminal surface to coating onthe luminal surface of the device is 75:25. In some embodiments, theratio of coating on the abluminal surface to coating on the luminalsurface of the device is 70:30. In some embodiments, the ratio ofcoating on the abluminal surface to coating on the luminal surface ofthe device is 60:40.

In some embodiments, the stent is a coronary stent, a vascular stent, aperipheral stent, billiarty stent, and intercranial stent.

EXAMPLES

The following examples are provided to illustrate selected embodiments.They should not be considered as limiting the scope of the invention,but merely as being illustrative and representative thereof. For eachexample listed below, multiple analytical techniques may be provided.Any single technique of the multiple techniques listed may be sufficientto show the parameter and/or characteristic being tested, or anycombination of techniques may be used to show such parameter and/orcharacteristic. Those skilled in the art will be familiar with a widerange of analytical techniques for the characterization of drug/polymercoatings. Techniques presented here, but not limited to, may be used toadditionally and/or alternatively characterize specific properties ofthe coatings with variations and adjustments employed which would beobvious to those skilled in the art.

Sample Preparation

Generally speaking, coatings on stents, on coupons, or samples preparedfor in-vivo models are prepared as below. Nevertheless, modificationsfor a given analytical method are presented within the examples shown,and/or would be obvious to one having skill in the art. Thus, numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein and examples provided may be employed in practicing the inventionand showing the parameters and/or characteristics described.

Coatings on Stents

Coated stents as described herein and/or made by a method disclosedherein are prepared. In some examples, the coated stents have a targetedthickness of ˜15 microns (˜5 microns of active agent). In some examples,the coating process is PDPDP (Polymer, sinter, Drug, Polymer, sinter,Drug, Polymer, sinter) using deposition of drug in dry powder form anddeposition of polymer particles by RESS methods and equipment describedherein. In the illustrations below, resulting coated stents may have a3-layer coating comprising polymer (for example, PLGA) in the firstlayer, drug (for example, rapamycin) in a second layer and polymer inthe third layer, where a portion of the third layer is substantiallydrug free (e.g. a sub-layer within the third layer having a thicknessequal to a fraction of the thickness of the third layer). As describedlayer, the middle layer (or drug layer) may be overlapping with one orboth first (polymer) and third (polymer) layer. The overlap between thedrug layer and the polymer layers is defined by extension of polymermaterial into physical space largely occupied by the drug. The overlapbetween the drug and polymer layers may relate to partial packing of thedrug particles during the formation of the drug layer. When crystal drugparticles are deposited on top of the first polymer layer, voids and orgaps may remain between dry crystal particles. The voids and gaps areavailable to be occupied by particles deposited during the formation ofthe third (polymer) layer. Some of the particles from the third(polymer) layer may rest in the vicinity of drug particles in the second(drug) layer. When the sintering step is completed for the third(polymer) layer, the third polymer layer particles fuse to form acontinuous film that forms the third (polymer) layer. In someembodiments, the third (polymer) layer however will have a portion alongthe longitudinal axis of the stent whereby the portion is free ofcontacts between polymer material and drug particles. The portion of thethird layer that is substantially of contact with drug particles can beas thin as 1 nanometer.

Polymer-coated stents having coatings comprising polymer but no drug aremade by a method disclosed herein and are prepared having a targetedthickness of, for example, 5 microns. An example coating process isPPP(PLGA, sinter, PLGA, sinter, PLGA, sinter) using RESS methods andequipment described herein. These polymer-coated stents may be used ascontrol samples in some of the examples, infra.

In some examples, the stents are made of a cobalt-chromium alloy and are5 to 50 mm in length, preferably 10-20 mm in length, with struts ofthickness between 20 and 100 microns, preferably 50-70 microns,measuring from an abluminal surface to a luminal surface, or measuringfrom a side wall to a side wall. In some examples, the stent may be cutlengthwise and opened to lay flat be visualized and/or assayed using theparticular analytical technique provided.

The coating may be removed (for example, for analysis of a coating bandand/or coating on a strut, and/or coating on the abluminal surface of aflattened stent) by scraping the coating off using a scalpel, knife orother sharp tool. This coating may be sliced into sections which may beturned 90 degrees and visualized using the surface compositiontechniques presented herein or other techniques known in the art forsurface composition analysis (or other characteristics, such ascrystallinity, for example). In this way, what was an analysis ofcoating composition through a depth when the coating was on the stent oras removed from the stent (i.e. a depth from the abluminal surface ofthe coating to the surface of the removed coating that once contactedthe strut or a portion thereof), becomes a surface analysis of thecoating which can, for example, show the layers in the slice of coating,at much higher resolution. Coating removed from the stent may be treatedthe same way, and assayed, visualized, and/or characterized as presentedherein using the techniques described and/or other techniques known to aperson of skill in the art.

Coatings on Coupons

In some examples, samples comprise coupons of glass, metal, e.g.cobalt-chromium, or another substance that are prepared with coatings asdescribed herein, with a plurality of layers as described herein, and/ormade by a method disclosed herein. In some examples, the coatingscomprise polymer. In some examples, the coatings comprise polymer andactive agent. In some examples, the coated coupons are prepared having atargeted thickness of ˜10 microns (with 5 microns of active agent), andhave coating layers as described for the coated stent samples, infra.

Sample Preparation for In-Vivo Models

Devices comprising stents having coatings disclosed herein are implantedin the porcine coronary arteries of pigs (domestic swine, juvenile farmpigs, or Yucatan miniature swine). Porcine coronary stenting isexploited herein since such model yields results that are comparable toother investigations assaying neointimal hyperplasia in human subjects.The stents are expanded to a 1:1.1 balloon:artery ratio. At multipletime points, animals are euthanized (e.g. t=1 day, 7 days, 14 days, 21days, and 28 days), the stents are explanted, and assayed.

Devices comprising stents having coatings disclosed herein alternativelyare implanted in the common iliac arteries of New Zealand white rabbits.The stents are expanded to a 1:1.1 balloon:artery ratio. At multipletime points, animals are euthanized (e.g., t=1 day, 7 days, 14 days, 21days, and 28 days), the stents are explanted, and assayed.

Example 1

This example illustrates embodiments that provide a coated coronarystent, comprising: a stent framework and a rapamycin-polymer coatingwherein at least part of rapamycin is in crystalline form and therapamycin-polymer coating comprises one or more resorbable polymers.

In these experiments two different polymers were employed:

-   -   Polymer A: −50:50 PLGA-Ester End Group, MW˜19 kD, degradation        rate ˜1-2 months    -   Polymer B: −50:50 PLGA-Carboxylate End Group, MW˜10 kD,        degradation rate ˜28 days

Metal stents were coated as follows:

-   -   AS1: Polymer A/Rapamycin/Polymer A/Rapamycin/Polymer A    -   AS2: Polymer A/Rapamycin/Polymer A/Rapamycin/Polymer B    -   AS1 (B) or AS1(213): Polymer B/Rapamycin/Polymer        B/Rapamycin/Polymer B    -   AS1b: Polymer A/Rapamycin/Polymer A/Rapamycin/Polymer A    -   AS2b: Polymer A/Rapamycin/Polymer A/Rapamycin/Polymer B

Example 2 Crystallinity

The presence and or quantification of the Active agent crystallinity canbe determined from a number of characterization methods known in theart, but not limited to, XRPD, vibrational spectroscopy (FTIR, NIR,Raman), polarized optical microscopy, calorimetry, thermal analysis andsolid-state NMR.

X-Ray Diffraction to Determine the Presence and/or Quantification ofActive Agent Crystallinity

Active agent and polymer coated proxy substrates are prepared using 316Lstainless steel coupons for X-ray powder diffraction (XRPD) measurementsto determine the presence of crystallinity of the active agent. Thecoating on the coupons is equivalent to the coating on the stentsdescribed herein. Coupons of other materials described herein, such ascobalt-chromium alloys, may be similarly prepared and tested. Likewise,substrates such as stents, or other medical devices described herein maybe prepared and tested. Where a coated stent is tested, the stent may becut lengthwise and opened to lay flat in a sample holder.

For example XRPD analyses are performed using an X-ray powderdiffractometer (for example, a Bruker D8 Advance X-ray diffractometer)using Cu Kα radiation. Diffractograms are typically collected between 2and 40 degrees 2 theta. Where required low background XRPD sampleholders are employed to minimize background noise.

The diffractograms of the deposited active agent are compared withdiffractograms of known crystallized active agents, for examplemicronized crystalline sirolimus in powder form. XRPD patterns ofcrystalline forms show strong diffraction peaks whereas amorphous showdiffuse and non-distinct patterns. Crystallinity is shown in arbitraryIntensity units.

A related analytical technique which may also be used to providecrystallinity detection is wide angle scattering of radiation (e.g.;Wide Anle X-ray Scattering or WAXS), for example, as described in F.Unger, et al., “Poly(ethylene carbonate): A thermoelastic andbiodegradable biomaterial for drug eluting stent coatings?” Journal ofControlled Release, Volume 117, Issue 3, 312-321 (2007) for which thetechnique and variations of the technique specific to a particularsample would be obvious to one of skill in the art.

Raman Spectroscopy

Raman spectroscopy, a vibrational spectroscopy technique, can be useful,for example, in chemical identification, characterization of molecularstructures, effects of bonding, identification of solid state form,environment and stress on a sample. Raman spectra can be collected froma very small volume (<1 μm³); these spectra allow the identification ofspecies present in that volume. Spatially resolved chemical information,by mapping or imaging, terms often used interchangeably, can be achievedby Raman microscopy.

Raman spectroscopy and other analytical techniques such as described inBalss, et al., “Quantitative spatial distribution of sirolimus andpolymers in drug-eluting stents using confocal Raman microscopy” J. ofBiomedical Materials Research Part A, 258-270 (2007), incorporated inits entirety herein by reference, and/or described in Belu et al.,“Three-Dimensional Compositional Analysis of Drug Eluting Stent CoatingsUsing Cluster Secondary Ion Mass Spectroscopy” Anal. Chem. 80: 624-632(2008) incorporated herein in its entirety by reference may be used.

For example, to test a sample using Raman microscopy and in particularconfocal Raman microscopy, it is understood that to get appropriateRaman high resolution spectra sufficient acquisition time, laser power,laser wavelength, sample step size and microscope objective need to beoptimized. For example a sample (a coated stent) is prepared asdescribed herein. Alternatively, a coated coupon could be tested in thismethod. Maps are taken on the coating using Raman microscopy. A WITecCRM 200 scanning confocal Raman microscope using a Nd:YAG laser at 532nm is applied in the Raman imaging mode. The laser light is focused uponthe sample using a 10× dry objective (numerical aperture 0.90), and thefinely focused laser spot is scanned into the sample. As the laser scansthe sample, over each 0.33 micron interval a Raman spectrum with highsignal to noise is collected using 0.3 seconds of integration time. Eachconfocal cross-sectional image of the coatings displays a region 70 μmwide by 10 μm deep, and results from the gathering of 6300 spectra witha total imaging time of 32 min.

Multivariate analysis using reference spectra from samples of rapamycin(amorphous and crystalline) and polymer are used to deconvolve thespectral data sets, to provide chemical maps of the distribution.

Infrared (IR) Spectroscopy for In-Vitro Testing

Infrared (IR) Spectroscopy such as FTIR and ATR-IR are well utilizedtechniques that can be applied to show, for example, the quantitativedrug content, the distribution of the drug in the sample coating, thequantitative polymer content in the coating, and the distribution ofpolymer in the coating. Infrared (IR) Spectroscopy such as FTIR andATR-IR can similarly be used to show, for example, drug crystallinity.The following table (Table 1) lists the typical IR materials for variousapplications. These IR materials are used for IR windows, diluents orATR crystals.

TABLE 1 MATERIAL NACL KBR CSI AGCL GE ZNSE DIAMOND Transmission40,000~625 40,000~400 40,000~200 25,000~360 5,500~625 20,000~45440,000~2,500 & range (cm−1) 1667-33 Water sol 35.7 53.5 44.4 Insol.Insol. Insol. Insol. (g/100 g, 25 C.) Attacking Wet Wet Wet AmmoniumH2SO4, Acids, K2Cr2Os, materials Solvents Solvents Solvents Salts aquaregin strong conc. alkalies, H2SO4 chlorinated solvents

In one test, a coupon of crystalline ZnSe is coated by the processesdescribed herein, creating a PDPDP (Polymer, Drug, Polymer, Drug,Polymer) layered coating that is about 10 microns thick. The coatedcoupon is analyzed using FTIR. The resulting spectrum shows crystallinedrug as determined by comparison to the spectrum obtained for thecrystalline form of a drug standard (i.e. a reference spectrum).

Differential Scanning Calorimetry (DSC)

DSC can provide qualitative evidence of the crystallinity of the drug(e.g. rapamycin) using standard DSC techniques obvious to one of skilledin the art. Crystalline melt can be shown using this analytical method(e.g. rapamycin crystalline melting—at about 185 decrees C to 200degrees C., and having a heat of fusion at or about 46.8 J/g). The heatof fusion decreases with the percent crystallinity. Thus, the degree ofcrystallinity could be determined relative to a pure sample, or versus acalibration curve created from a sample of amorphous drug spiked andtested by DSC with known amounts of crystalline drug. Presence (atleast) of crystalline drug on a stent could be measured by removing(scraping or stripping) some drug from the stent and testing the coatingusing the DSC equipment for determining the melting temperature and theheat of fusion of the sample as compared to a known standard and/orstandard curve.

Example 3 Determination of Bioabsorbability/Bioresorbability/DissolutionRate of a Polymer Coating a Device Gel Permeation Chromatography In-vivoWeight Loss Determination

Standard methods known in the art can be applied to determine polymerweight loss, for example gel permeation chromatography and otheranalytical techniques such as described in jackson et al.,“Characterization of perivascular poly(lactic-co-glycolic acid) filmscontaining paclitaxel” Int. J. of Pharmaceutics, 283:97-109 (2004),incorporated in its entirety herein by reference.

For example rabbit in vivo models as described above are euthanized atmultiple time points (t=1 day, 2 days, 4 days, 7 days, 14 days, 21 days,28 days, 35 days n=5 per time point). Alternatively, pig in vivo modelsas described above are euthanized at multiple time points (t=1 day, 2days, 4 days, 7 days, 14 days, 21 days, 28 days, 35 days n=5 per timepoint). The stents are explanted, and dried down at 30° C. under astream of gas to complete dryness. A stent that has not been implantedin the animal is used as a control for no loss of polymer.

The remaining polymer on the explanted stents is removed using asolubilizing solvent (for example chloroform). The solutions containingthe released polymers for each time point are filtered. Subsequent GPCanalysis is used for quantification of the amount of polymer remainingin the stent at each explant time point. The system, for example,comprises a Shimadzu LC-10 AD HPLC pump, a Shimadzu RID-6A refractiveindex detector coupled to a 50 Å Hewlett Packard Pl-Gel column. Thepolymer components are detected by refractive index detection and thepeak areas are used to determine the amount of polymer remaining in thestents at the explant time point. A calibration graph of log molecularweight versus retention time is established for the 50 A Pl-Gel columnusing polystyrene standards with molecular weights of 300, 600, 1.4 k, 9k, 20 k, and 30 k g/mol. The decreases in the polymer peak areas on thesubsequent time points of the study are expressed as weight percentagesrelative to the 0 day stent.

Gel Permeation Chromatography In-Vitro testing

Gel Permeation Chromatography (GPC) can also be used to quantify thebioabsorbability/bioresorbability, dissolution rate, and/orbiodegrability of the polymer coating. The in vitro assay is adegradation test where the concentration and molecular weights of thepolymers can be assessed when released from the stents in an aqueoussolution that mimics physiological surroundings. See for example,Jackson et al., “Characterization of perivascularpoly(lactic-co-glycolic acid) films containing paclitaxel” Int. J. ofPharmaceutics, 283:97-109 (2004), incorporated in its entirety herein byreference.

For example Stents (n=15) described herein are expanded and then placedin a solution of 1.5 ml solution of phosphate buffered saline (pH=7.4)with 0.05% wt of Tween20, or in the alternative 10 mM Tris, 0.4 wt. %SDS, pH 7.4, in a 37° C. bath with bath rotation at 70 rpm.Alternatively, a coated coupon could be tested in this method. Thesolution is then collected at the following time points: 0 min., 15min., 30 min., 1 hr, 2 hr, 4 hr, 6 hr, 8 hr, 12 hr, 16 hr, 20 hr, 24 hr,30 hr, 36 hr, 48 hr, and daily up to 70 days, for example. The solutionis replaced at least at each time point, and/or periodically (e.g. everyfour hours, daily, weekly, or longer for later time points) to preventsaturation, the removed solution is collected, saved, and assayed. Thesolutions containing the released polymers for each time point arefiltered to reduce clogging the GPC system. For time points over 4hours, the multiple collected solutions are pooled together for liquidextraction.

1 ml Chloroform is added to the phosphate buffered saline solutions andshaken to extract the released polymers from the aqueous phase. Thechloroform phase is then collected for assay via GPC.

The system comprises a Shimadzu LC-10 AD HPLC pump, a Shimadzu RID-6Arefractive index (RI) detector coupled to a 50 Å Hewlett Packard Pl-Gelcolumn. The mobile phase is chloroform with a flow rate of 1 mL/min. Theinjection volume of the polymer sample is 100 μL of a polymerconcentration. The samples are run for 20 minutes at an ambienttemperature.

For determination of the released polymer concentrations at each timepoint, quantitative calibration graphs are first made using solutionscontaining known concentrations of each polymer in chloroform. Stocksolutions containing each polymer in 0-5 mg/ml concentration range arefirst analyzed by GPC and peak areas are used to create separatecalibration curves for each polymer.

For polymer degradation studies, a calibration graph of log molecularweight versus retention time is established for a 50 Å Pl-Gel column(Hewlett Packard) using polystyrene standards with molecular weights of300, 600, 1.4 k, 9 k, 20 k, and 30 k g/mol. In the alternative, a Multiangle light scattering (MALS) detector may be fitted to directly assessthe molecular weight of the polymers without the need of polystyrenestandards.

To perform an accelerated in-vitro dissolution of the bioresorbablepolymers, a protocol is adapted from ISO Standard 13781 “Poly(L-lactide)resides and fabricated an accelerated froms for surgical implants—invitro degradation testing” (1997), incorporated in its entirety hereinby reference. Briefly, elution buffer comprising 18% v/v of a stocksolution of 0.067 mol/L KH₂PO₄ and 82% v/v of a stock solution of 0.067mol/L Na₂HPO₄ with a pH of 7.4 is used. Stents described herein areexpanded and then placed in 1.5 ml solution of this accelerated elutionin a 70° C. bath with rotation at 70 rpm. The solutions are thencollected at the following time points: 0 min., 15 min., 30 min., 1 hr,2 hr, 4 hr, 6 hr, 8 hr, 12 hr, 16 hr, 20 hr, 24 hr, 30 hr, 36 hr and 48hr. Fresh accelerated elution buffer are added periodically every twohours to replace the incubated buffers that are collected and saved inorder to prevent saturation. The solutions containing the releasedpolymers for each time point are filtered to reduce clogging the GPCsystem. For time points over 2 hours, the multiple collected solutionsare pooled together for liquid extraction by chloroform. Chloroformextraction and GPC analysis is performed in the manner described above.

Scanning Electron Microscopy (SEM) with Focused Ion Beam (FIB) MillingIn-Vitro Testing

Focused ion beam FIB is a tool that allows precise site-specificsectioning, milling and depositing of materials. FIB can be used inconjunction with SEM, at ambient or cryo conditions, to produce in-situsectioning followed by high-resolution imaging. FIB-SEM can produce across-sectional image of the polymer layers on the stent. The image canbe used to quantitate the thickness of the layers to reveal rate ofbioresorbability of single or multiple polymers as well as show whetherthere is uniformity of the layer thickness at manufacture and at timepoints after stenting (or after in-vitro elution at various timepoints).

For example, testing is performed at multiple time points. Stents areremoved from the elution media and dried, the dried stent is visualizedusing FIB-SEM for changes in the coating. Alternatively, a coated couponcould be tested in this method.

Stents (n=15) described herein are expanded and then placed in 1.5 mlsolution of phosphate buffered saline (pH=7.4) with 0.05% wt of Tween20in a 37° C. bath with bath rotation at 70 rpm. Alternatively, a coatedcoupon could be tested in this method. The phosphate buffered salinesolution is periodically replaced with fresh solution at each time pointand/or every four hours to prevent saturation. The stents are collectedat the following time points: 30 min, 1 hr, 2 hr, 4 hr, 6 hr, 8, hr, 12hr, 16 hr, 20 hr, 24 hr, 30 hr, 36 hr, 48 hr, 60 h and 72 h. The stentsare dried down at 30° C. under a stream of gas to complete dryness. Astent that not been subjected to these conditions is used as a t=0control.

A FEI Dual Beam Strata 235 FIB/SEM system is a combination of a finelyfocused Ga ion beam (FIB) accelerated by 30 kV with a field emissionelectron beam in a scanning electron microscope instrument and is usedfor imaging and sectioning the stents. Both beams focus at the samepoint of the sample with a probe diameter less than 10 nm. The FIB canalso produce thinned down sections for TEM analysis.

To prevent damaging the surface of the stent with incident ions, a Ptcoating is first deposited via electron beam assisted deposition and ionbeam deposition prior to FIB sectioning. For FIB sectioning, the Ga ionbeam is accelerated to 30 kV and the sectioning process is about 2 h induration. Completion of the FIB sectioning allows one to observe andquantify by SEM the thickness of the polymer layers that are left on thestent as they are absorbed.

Raman Spectroscopy In-Vitro Testing

As discussed in example 2, Raman spectroscopy can be applied tocharacterize the chemical structure and relative concentrations of drugand polymer coatings. This can also be applied to characterize in-vitrotested polymer coatings on stents or other substrates.

For example, confocal Raman Spectroscopy/microscopy can be used tocharacterize the relative drug to polymer ratio at the outer 1 μm of thecoated surface as a function of time exposed to elution media. Inaddition confocal Raman x-z or z (maps or line scans) microscopy can beapplied to characterize the relative drug to polymer ratio as a functionof depth at time t after exposure to elution media.

For example a sample (a coated stent) is prepared as described hereinand placed in elution media (e.g., 10 mM tris(hydroxymethyl)aminomethane(Tris), 0.4 wt. % Sodium dodecyl sulphate (SDS), pH 7.4 or 1.5 mlsolution of phosphate buffered saline (pH=7.4) with 0.05% wt of Tween20)in a 37° C. bath with bath rotation at 70 rpm. Confocal Raman Images aretaken on the coating before elution. At least four elution time pointswithin a 48 day interval, (e.g. 0 min., 15 min., 30 min., 1 hr, 2 hr, 4hr, 6 hr, 8, hr, 12 hr, 16 hr, 20 hr, 24 hr, 30 hr, 36 hr and 48 hr) thesample is removed from the elution, and dried (for example, in a streamof nitrogen). The dried stent is visualized using Raman Spectroscopy forchanges in coating. Alternatively, a coated coupon could be tested inthis method. After analysis, each is returned to the buffer for furtherelution.

Raman spectroscopy and other analytical techniques such as described inBalss, et al., “Quantitative spatial distribution of sirolimus andpolymers in drug-eluting stents using confocal Raman microscopy” J. ofBiomedical Materials Research Part A, 258-270 (2007), incorporated inits entirety herein by reference, and/or described in Belu et al.,“Three-Dimensional Compositional Analysis of Drug Eluting Stent CoatingsUsing Cluster Secondary Ion Mass Spectroscopy” Anal. Chem. 80: 624-632(2008) incorporated herein in its entirety by reference may be used.

For example a WITec CRM 200 scanning confocal Raman microscope using aNd:YAG laser at 532 nm is applied in the Raman imaging mode to generatean x-z map. The sample is placed upon a piezoelectrically driven table,the laser light is focused upon the sample using a 10× dry objective(numerical aperture 0.90), and the finely focused laser spot is scannedinto the sample. As the laser scans the sample, over each 0.33 microninterval a Raman spectrum with high signal to noise is collected using0.3 Seconds of integration time. Each confocal cross-sectional image ofthe coatings displays a region 70 μm wide by 10 μm deep, and resultsfrom the gathering of 6300 spectra with a total imaging time of 32 min.

SEM-In-Vitro Testing

Testing is performed at multiple time points (e.g. 0 min., 15 min., 30min., 1 hr, 2 hr, 4 hr, 6 hr, 8, hr, 12 hr, 16 hr, 20 hr, 24 hr, 30 hr,36 hr and 48 hr). Stents are removed from the elution media (describedsupra) and dried at these time points. The dried stent is visualizedusing SEM for changes in coating.

For example the samples are observed by SEM using a Hitachi S-4800 withan accelerating voltage of 800V. Various magnifications are used toevaluate the coating integrity, especially at high strain regions.Change in coating over time is evaluated to visualize the bioabsorptionof the polymer over time.

X-ray photoelectron spectroscopy (XPS)—In-Vitro Testing

XPS can be used to quantitatively determine elemental species andchemical bonding environments at the outer 5-10 nm of sample surface.The technique can be operated in spectroscopy or imaging mode. Whencombined with a sputtering source, XPS can be utilized to give depthprofiling chemical characterization.

XPS testing can be used to characterize the drug to polymer ratio at thevery surface of the coating of a sample. Additionally XPS testing can berun in time lapse to detect changes in composition. Thus, in one test,samples are tested using XPS at multiple time points (e.g. 0 min., 15min., 30 min., 1 hr, 2 hr, 4 hr, 6 hr, 8, hr, 12 hr, 16 hr, 20 hr, 24hr, 30 hr, 36 hr and 48 hr). Stents are removed from the elution media(e.g., 10 mM Tris, 0.4 wt. % SDS, pH 7.4 or 1.5 ml solution of phosphatebuffered saline (pH=7.4) with 0.05% wt of Tween20) in a 37° C. bath withrotation at 70 rpm and dried at these time points.

XPS (ESCA) and other analytical techniques such as described in Belu etal, “Three-Dimensional Compositional Analysis of Drug Eluting StentCoatings Using Cluster Secondary Ion Mass Spectroscopy” Anal. Chem. 80:624-632 (2008) incorporated herein in its entirety by reference may beused.

For example, XPS analysis is performed using a Physical ElectronicsQuantum 2000 Scanning ESCA. The monochromatic Al Kα source is operatedat 15 kV with a power of 4.5 W. The analysis is performed at a 45° takeoff angle. Three measurements are taken along the length of each stentwith the analysis area 20 microns in diameter. Low energy electron andAr⁺ ion floods are used for charge compensation.

Time of Flight Secondary Ion Mass Spectrometery (TOF-SIMS)

TOF-SIMS can be used to determine molecular species at the outer 1-2 nmof sample surface when operated under static conditions. The techniquecan be operated in spectroscopy or imaging mode at high spatialresolution. When operated under dynamic experimental conditions, knownin the art, depth profiling chemical characterization can be achieved.

TOF-SIMS testing can be used to characterize the presence of polymer andor drug at uppermost surface of the coating of a sample. AdditionallyTOF-SIMS testing can be run in time lapse to detect changes incomposition. Thus, in one test, samples are tested using TOF-SIMS atmultiple time points (e.g., 0 min., 15 min., 30 min., 1 hr, 2 hr, 4 hr,6 hr, 8, hr, 12 hr, 16 hr, 20 hr, 24 hr, 30 hr, 36 hr and 48 hr). Stentsare removed from the elution media (e.g. 10 mM Tris, 0.4 wt. % SDS, pH7.4 or 1.5 ml solution of phosphate buffered saline (pH=7.4) with 0.05%wt of Tween20) in a 37° C. bath with rotation at 70 rpm and dried atthese time points.

For example, to analyze the uppermost surface only, static conditions(for example a ToF-SIMS IV (IonToF, Munster)) using a 25 Kv Bi⁺⁺ primaryion source maintained below 10¹² ions per cm² is used. Where necessary alow energy electron flood gun (0.6 nA DC) is used to charge compensateinsulating samples.

Cluster Secondary Ion Mass Spectrometry, may be employed for depthprofiling as described Belu et al., “Three-Dimensional CompositionalAnalysis of Drug Eluting Stent Coatings Using Cluster Secondary Ion MassSpectroscopy” Anal. Chem. 80: 624-632 (2008) incorporated herein in itsentirety by reference.

For example, a stent as described herein is obtained. The stent isprepared for SIMS analysis by cutting it longitudinally and opening itup with tweezers. The stent is then pressed into multiple layers ofindium foil with the outer diameter facing outward.

TOF-SIMS depth profiling experiments are performed using an Ion-TOF IVinstrument equipped with both Bi and SF5+ primary ion beam clustersources. Sputter depth profiling is performed in the dual-beam mode,while preserving the chemical integrity of the sample. For example, theanalysis source is a pulsed, 25-keV bismuth cluster ion source, whichbombarded the surface at an incident angle of 45° to the surface normal.The target current is maintained at ˜0.3 pÅ (+10%) pulsed current with araster size of 200 micron×200 micron for all experiments. Both positiveand negative secondary ions are extracted from the sample into areflectron-type time-of-flight mass spectrometer. The secondary ions arethen detected by a microchannel plate detector with a post-accelerationenergy of 10 kV. A low-energy electron flood gun is utilized for chargeneutralization in the analysis mode.

The sputter source used is a 5-keV SF5+ cluster source also operated atan incident angle of 45° to the surface normal. For thin model sampleson Si, the SF5+ current is maintained at ˜2.7 nÅ with a 750 micron×750micron raster. For the thick samples on coupons and for the samples onstents, the current is maintained at 6 nA with a 500 micron×500 micronraster. All primary beam currents are measured with a Faraday cup bothprior to and after depth profiling.

All depth profiles are acquired in the noninterlaced mode with a 5-mspause between sputtering and analysis. Each spectrum is averaged over a7.37 second time period. The analysis is immediately followed by 15seconds of SF₅ ⁺ sputtering. For depth profiles of the surface andsubsurface regions only, the sputtering time was decreased to 1 secondfor the 5% active agent sample and 2 seconds for both the 25% and 50%active agent samples. Temperature-controlled depth profiles are obtainedusing a variable-temperature stage with Eurotherm Controls temperaturecontroller and IPSG V3.08 software. Samples are first placed into theanalysis chamber at room temperature. The samples are brought to thedesired temperature under ultra high-vacuum conditions and are allowedto stabilize for 1 minute prior to analysis. All depth profilingexperiments are performed at −100 degrees C. and 25 degrees C.

Infrared (IR) Spectroscopy for In-Vitro Testing

Infrared (IR) Spectroscopy such as, but not limited to, FTIR, ATR-IR andmicro ATR-IR are well utilized techniques that can be applied to showthe quantitative polymer content in the coating, and the distribution ofpolymer in the coating.

For example using FTIR, a coupon of crystalline ZnSe is coated by theprocesses described herein, creating a PDPDP (Polymer, Drug, Polymer,Drug, Polymer) layered coating that is about 10 microns thick. At time=0and at least four elution time points within a 48 day interval (e.g., 0min., 15 min., 30 min., 1 hr, 2 hr, 4 hr, 6 hr, 8, hr, 12 hr, 16 hr, 20hr, 24 hr, 30 hr, 36 hr and 48 hr), the sample (coated crystal) wastested by FTIR for polymer content. The sample was placed in an elutionmedia (e.g. 10 mM Tris, 0.4 wt. % SDS, pH 7.4 or 1.5 ml solution ofphosphate buffered saline (pH=7.4) with 0.05% wt of Tween20) in a 37° C.bath with bath rotation at 70 rpm and at each time point, the sample isremoved from the elution media and dried (e.g. in a stream of nitrogen).FTIR spectrometry was used to quantify the polymer on the sample. Afteranalysis, each is returned to the buffer for further elution.

In another example using FTIR, sample elution media at each time pointwas tested for polymer content. In this example, a coated stent wasprepared that was coated by the processes described herein, creating aPDPDP (Polymer, Drug, Polymer, Drug, Polymer) layered coating that isabout 10 microns thick. The coated stent was placed in an elution media(e.g. 10 mM Tris, 0.4 wt. % SDS, pH 7.4 or 1.5 ml solution of phosphatebuffered saline (pH=7.4) with 0.05% wt of Tween20) in a 37° C. bath withrotation at 70 rpm. and at each time point (e.g., 0 min., 15 min., 30min., 1 hr, 2 hr, 4 hr, 6 hr, 8, hr, 12 hr, 16 hr, 20 hr, 24 hr, 30 hr,36 hr and 48 hr), a sample of the elution media is removed and driedonto a crystalline ZnSe window (e.g. in a stream of nitrogen). At eachelution time point, the sample elution media was tested by FTIR forpolymer content.

Atomic Force Microscopy (AFM)

AFM is a high resolution surface characterization technique. AFM is usedin the art to provide topographical imaging, in addition when employedin Tapping Mode™ can image material and or chemical properties of thesurface. The technique can be used under ambient, solution, humidifiedor temperature controlled conditions. Other modes of operation are wellknown and can be readily employed here by those skilled in the art. TheAFM topography images can be run in time-lapse to characterize thesurface as a function of elution time. Three-dimensionally renderedimages show the surface of a coated stent, which can show holes or voidsof the coating which may occur as the polymer is absorbed and the drugis eluted over time.

A stent as described herein is obtained. AFM is used to determine thedrug polymer distribution. AFM may be employed as described in Ranade etal., “Physical characterization of controlled release of paclitaxel fromthe TAXUS Express2 drug-eluting stent” J. Biomed. Mater. Res.71(4):625-634 (2004) incorporated herein in its entirety by reference.

For example a multi-mode AFM (Digital Instruments/Veeco Metrology, SantaBarbara, Calif.) controlled with Nanoscope IIIa and NanoScope Extenderelectronics is used. Samples are examined in the dry state using AFMbefore elution of the drug (e.g. rapamycin). Samples are also examinedat select time points through a elution period (e.g. 48 hours) by usingan AFM probe-tip and flow-through stage built to permit analysis of wetsamples. The wet samples are examined in the presence of the sameelution medium used for in-vitro kinetic drug release analysis (e.g.PBS-Tween20, or 10 mM Tris, 0.4 wt. % SDS, pH 7.4). Saturation of thesolution is prevented by frequent exchanges of the release medium withseveral volumes of fresh medium. TappingMode™ AFM imaging may be used toshow topography (a real-space projection of the coating surfacemicrostructure) and phase-angle changes of the AFM over the sample areato contrast differences in the material and physical structure.

Nano X-Ray Computer Tomography

Another technique that may be used to view the physical structure of adevice in 3-D is Nano X-Ray Computer Tomography (e.g. such as made bySkyScan), which could be used in an elution test and/or bioabsorbabilitytest, as described herein to show the physical structure of the coatingremaining on stents at each time point, as compared to a scan prior toelution/bioabsorbtion.

pH Testing

The bioabsorbability of PLGA of a coated stent can be shown by testingthe pH of an elution media (EtOH/PBS, for example) in which the coatedstent is placed. Over time, a bioabsorbable PLGA coated stent (with orwithout the drug) will show a decreased pH until the PLGA is fullybioabsorbed by the elution media.

A test was performed using stents coated with PLGA alone, stents coatedwith PLGA and rapamycin, PLGA films, and PLGA films containingrapamycin. The samples were put in elution media of 20% EtOH/PBS at 37°C. The elution media was tested at multiple intervals from 0 to 48 days.In FIGS. 1, 2 and 3, stents having coatings as provided herein weretested for pH over time according to this method. FIG. 4 shows resultsof the PLGA films (with and without rapamycin) tested according to thismethod. Control elution media was run in triplicate alongside thesamples, and the results of this pH testing was averaged and ispresented as “Control AVE” in each of the FIGS. 1-4.

In FIG. 2, the “30D2Rapa Stents ave” line represents a stent havingcoating according to AS1(213) of Example 1 (PDPDP) with Polymer B (50:50PLGA-Carboxylate end group, MW ˜10 kD) and rapamycin, where the coatingwas removed from the stent and tested in triplicate for pH changes overtime in the elution media, the average of which is presented. The “30D2Stents ave” line represents a stent having coating of only Polymer B(50:50 PLGA-Carboxylate end group, MW ˜10 kD) (no rapamycin), where thecoating was removed from the stent and tested in triplicate for pHchanges over time in the elution media, the average of which ispresented.

In FIG. 1, the “60DRapa Stents ave” line represents a stent havingcoating according to AS1 of Example 1 (PDPDP) with Polymer A (50:50PLGA-Ester end group, MW ˜19 kD) and rapamycin, where the coating wasremoved from the stent and tested in triplicate for pH changes over timein the elution media, the average of which is presented.

The “60D Stents ave” line represents a stent having coating of onlyPolymer A (50:50 PLGA-Ester end group, MW ˜19 kD) (no rapamycin), wherethe coating was removed from the stent and tested in triplicate for pHchanges over time in the elution media, the average of which ispresented.

In FIG. 3, the “85:15 Rapa Stents ave” line represents a stent havingcoating according to PDPDP with a PLGA comprising 85% lactic acid, 15%glycolic acid, and rapamycin, where the coating was removed from thestent and tested in triplicate for pH changes over time in the elutionmedia, the average of which is presented. The “85:15 Stents ave” linerepresents a stent having coating of only PLGA comprising 85% lacticacid, 15% glycolic acid (no rapamycin), where the coating was removedfrom the stent and tested in triplicate for pH changes over time in theelution media, the average of which is presented.

In FIG. 4, the “30D Ave” line represents a polymer film comprisingPolymer B (50:50 PLGA-Carboxylate end group, MW ˜10 kD) (no rapamycin),where the film was tested in triplicate for pH changes over time in theelution media, the average of which is presented. The “30D2 Ave” linealso represents a polymer film comprising Polymer B (50:50PLGA-Carboxylate end group, MW ˜10 kD) (no rapamycin), where the filmwas tested in triplicate for pH changes over time in the elution media,the average of which is presented. The “60D Ave” line represents apolymer film comprising Polymer A (50:50 PLGA-Ester end group, MW ˜19kD) (no rapamycin), where the film was tested in triplicate for pHchanges over time in the elution media, the average of which ispresented. The “85:15 Ave” line represents a polymer film comprisingPLGA comprising 85% lactic acid, 15% glycolic acid (no rapamycin), wherethe film was tested in triplicate for pH changes over time in theelution media, the average of which is presented. To create the polymerfilms in FIG. 4, the polymers were dissolved in methylene chloride, THF,and ethyl acetate. The films that were tested had the following averagethicknesses and masses, 30D-152.4 um, 12.0 mg; 30D2-127.0 um, 11.9 mg;60D-50.8 um, 12.4 mg; 85:15-127 um, 12.5 mg.

Example 4 Visualization of Polymer/Active Agent Layers Coating a DeviceRaman Spectroscopy

As discussed in example 2, Raman spectroscopy can be applied tocharacterize the chemical structure and relative concentrations of drugand polymer coatings. For example, confocal RamanSpectroscopy/microscopy can be used to characterize the relative drug topolymer ratio at the outer ˜1 μm of the coated surface. In additionconfocal Raman x-z or z (maps or line scans) microscopy can be appliedto characterize the relative drug to polymer ratio as a function ofdepth. Additionally cross-sectioned samples can be analysed. Ramanspectroscopy and other analytical techniques such as described in Balss,et al., “Quantitative spatial distribution of sirolimus and polymers indrug-eluting stents using confocal Raman microscopy” J. of BiomedicalMaterials Research Part A, 258-270 (2007), incorporated in its entiretyherein by reference, and/or described in Belu et al., “Three-DimensionalCompositional Analysis of Drug Eluting Stent Coatings Using ClusterSecondary Ion Mass Spectroscopy” Anal. Chem. 80: 624-632 (2008)incorporated herein in its entirety by reference may be used.

A sample (a coated stent) is prepared as described herein. Images aretaken on the coating using Raman Spectroscopy. Alternatively, a coatedcoupon could be tested in this method. To test a sample using Ramanmicroscopy and in particular confocal Raman microscopy, it is understoodthat to get appropriate Raman high resolution spectra sufficientacquisition time, laser power, laser wavelength, sample step size andmicroscope objective need to be optimized.

For example a WITec CRM 200 scanning confocal Raman microscope using aNd:YAG laser at 532 nm is applied in the Raman imaging mode to give x-zmaps. The sample is placed upon a piezoelectrically driven table, thelaser light is focused upon the sample using a 100× dry objective(numerical aperture 0.90), and the finely focused laser spot is scannedinto the sample. As the laser scans the sample, over each 0.33 microninterval a Raman spectrum with high signal to noise is collected using0.3 Seconds of integration time. Each confocal cross-sectional image ofthe coatings displays a region 70 μm wide by 10 μm deep, and resultsfrom the gathering of 6300 spectra with a total imaging time of 32 min.Multivariate analysis using reference spectra from samples of rapamycinand polymer are used to deconvolve the spectral data sets, to providechemical maps of the distribution.

In another test, spectral depth profiles (x-z maps) of samples areperformed with a CRM200 microscope system from WITec InstrumentsCorporation (Savoy, Ill.). The instrument is equipped with a Nd:YAGfrequency doubled laser (532 excitation), a single monochromator (Acton)employing a 600 groove/mm grating and a thermoelectrically cooled 1024by 128 pixel array CCD camera (Andor Technology). The microscope isequipped with appropriate collection optics that include a holographiclaser bandpass rejection filter (Kaiser Optical Systems Inc.) tominimize Rayleigh scatter into the monochromator. The Raman scatteredlight are collected with a 50 micron optical fiber. Using the “RamanSpectral Imaging” mode of the instrument, spectral images are obtainedby scanning the sample in the x, z direction with a piezo driven xyzscan stage and collecting a spectrum at every pixel. Typical integrationtimes are 0.3 s per pixel. The spectral images are 4800 total spectracorresponding to a physical scan dimension of 40 by 20 microns. Forpresentation of the confocal Raman data, images are generated based onunique properties of the spectra (i.e. integration of a Raman band, bandheight intensity, or band width). The microscope stage is modified witha custom-built sample holder that positioned and rotated the stentsaround their primary axis. The x direction is defined as the directionrunning parallel to the length of the stent and the z direction refersto the direction penetrating through the coating from the air-coating tothe coating-metal interface. Typical laser power is <10 mW on the samplestage. All experiments can be conducted with a plan a chromat objective,100×N_(A)=0.9 (Nikon).

Samples (n=5) comprising stents made of L605 (0.05-0.15% C, 1.00-2.00%Mn, maximum 0.040% Si, maximum 0.030% P, maximum 0.3% S, 19.00-21.00%Cr, 9.00-11.00% Ni, 14.00-16.00% W, 3.00% Fe, and Bal. Co) and havingcoatings as described herein and/or produced by methods described hereincan be analyzed. For each sample, three locations are selected along thestent length. The three locations are located within one-third portionsof the stents so that the entire length of the stent are represented inthe data. The stent is then rotated 180 degrees around the circumferenceand an additional three locations are sampled along the length. In eachcase, the data is collected from the strut portion of the stent. Sixrandom spatial locations are also profiled on coated coupon samples madeof L605 and having coatings as described herein and/or produced bymethods described herein. The Raman spectra of each individual componentpresent in the coatings are also collected for comparison and reference.Using the instrument software, the average spectra from the spectralimage data are calculated by selecting the spectral image pixels thatare exclusive to each layer. The average spectra are then exported intoGRAMS/AI v. 7.02 software (Thermo Galactic) and the appropriate Ramanbands are fit to a Voigt function. The band areas and shift positionsare recorded.

The pure component spectrum for each component of the coating (e.g.drug, polymer) are also collected at 532 and 785 nm excitation. The 785nm excitation spectra are collected with a confocal Raman microscope(WITec Instruments Corp. Savoy, Ill.) equipped with a 785 nm diodelaser, appropriate collection optics, and a back-illuminatedthermoelectriaclly cooled 1024×128 pixel array CCD camera optimized forvisible and infrared wavelengths (Andor Technology).

X-Ray Photoelectron Spectroscopy (XPS)

XPS can be used to quantitatively determine elemental species andchemical bonding environments at the outer 5-10 nm of sample surface.The technique can be operated in spectroscopy or imaging mode. Whencombined with a sputtering source XPS can be utilized to give depthprofiling chemical characterization. XPS (ESCA) and other analyticaltechniques such as described in Belu et al., “Three-DimensionalCompositional Analysis of Drug Eluting Stent Coatings Using ClusterSecondary Ion Mass Spectroscopy” Anal. Chem. 80: 624-632 (2008)incorporated herein in its entirety by reference may be used.

For example, in one test, a sample comprising a stent coated by methodsdescribed herein and/or a device as described herein is obtained. XPSanalysis is performed on a sample using a Physical Electronics Quantum2000 Scanning ESCA. The monochromatic Al Kα source is operated at 15 kVwith a power of 4.5 W. The analysis is done at a 45° take off angle.Three measurements are taken along the length of each sample with theanalysis area 20 microns in diameter. Low energy electron and Ar⁺ ionfloods are used for charge compensation.

Time of Flight Secondary Ion Mass Spectrometery (TOF-SIMS)

TOF-SIMS can be used to determine molecular species (drug and polymer)at the outer 1-2 nm of sample surface when operated under staticconditions. The technique can be operated in spectroscopy or imagingmode at high spatial resolution. Additionally cross-sectioned samplescan be analysed. When operated under dynamic experimental conditions,known in the art, depth profiling chemical characterization can beachieved.

For example, to analyze the uppermost surface only, static conditions(for example a ToF-SIMS IV (IonToF, Munster)) using a 25 Kv Bi+primaryion source maintained below 1012 ions per cm² is used. Where necessary alow energy electron flood gun (0.6 nA DC) is used to charge compensateinsulating samples.

Cluster Secondary Ion Mass Spectrometry, may be employed for depthprofiling as described Belu et al., “Three-Dimensional CompositionalAnalysis of Drug Eluting Stent Coatings Using Cluster Secondary Ion MassSpectroscopy” Anal. Chem. 80: 624-632 (2008) incorporated herein in itsentirety by reference.

For example, a stent as described herein is obtained. The stent isprepared for SIMS analysis by cutting it longitudinally and opening itup with tweezers. The stent is then pressed into multiple layers ofindium foil with the outer diameter facing outward.

TOF-SIMS depth profiling experiments are performed using an Ion-TOF IVinstrument equipped with both Bi and SF5+ primary ion beam clustersources. Sputter depth profiling is performed in the dual-beam mode,whilst preserving the chemical integrity of the sample. The analysissource is a pulsed, 25-keV bismuth cluster ion source, which bombardedthe surface at an incident angle of 45° to the surface normal. Thetarget current is maintained at 0.3 pA (+10%) pulsed current with araster size of 200 um×200 um for all experiments. Both positive andnegative secondary ions are extracted from the sample into areflectron-type time-of-flight mass spectrometer. The secondary ions arethen detected by a microchannel plate detector with a post-accelerationenergy of 10 kV. A low-energy electron flood gun is utilized for chargeneutralization in the analysis mode.

The sputter source used is a 5-keV SF5+ cluster source also operated atan incident angle of 45° to the surface normal. For thin model sampleson Si, the SF5+ current is maintained at ˜2.7 nA with a 750 um×750 umraster. For the thick samples on coupons and for the samples on stents,the current is maintained at 6 nA with a 500 um×500 um raster.

All primary beam currents are measured with a Faraday cup both prior toand after depth profiling.

All depth profiles are acquired in the noninterlaced mode with a 5-mspause between sputtering and analysis. Each spectrum is averaged over a7.37 second time period. The analysis is immediately followed by 15seconds of SF₅ ⁺ sputtering. For depth profiles of the surface andsubsurface regions only, the sputtering time was decreased to 1 secondfor the 5% active agent sample and 2 seconds for both the 25% and 50%active agent samples. Temperature-controlled depth profiles are obtainedusing a variable-temperature stage with Eurotherm Controls temperaturecontroller and IPSG V3.08 software. samples are first placed into theanalysis chamber at room temperature. The samples are brought to thedesired temperature under ultra high-vacuum conditions and are allowedto stabilize for 1 minute prior to analysis. All depth profilingexperiments are performed at −100 C and 25 C.

Atomic Force Microscopy (AFM)

AFM is a high resolution surface characterization technique. AFM is usedin the art to provide topographical imaging, in addition when employedin Tapping Mode™ can image material and or chemical properties of thesurface. Additionally cross-sectioned samples can be analyzed. Thetechnique can be used under ambient, solution, humidified or temperaturecontrolled conditions. Other modes of operation are well known and canbe readily employed here by those skilled in the art.

A stent as described herein is obtained. AFM is used to determine thestructure of the drug polymer layers. AFM may be employed as describedin Ranade et al., “Physical characterization of controlled release ofpaclitaxel from the TAXUS Express2 drug-eluting stent” J. Biomed. Mater.Res. 71(4):625-634 (2004) incorporated herein in its entirety byreference.

Polymer and drug morphologies, coating composition, at least may bedetermined using atomic force microscopy (AFM) analysis. A multi-modeAFM (Digital Instruments/Veeco Metrology, Santa Barbara, Calif.)controlled with Nanoscope IIIa and NanoScope Extender electronics isused. Samples are examined in the dry state using AFM before elution ofthe drug (e.g. rapamycin). Samples are also examined at select timepoints through a elution period (e.g. 48 hours) by using an AFMprobe-tip and flow-through stage built to permit analysis of wetsamples. The wet samples are examined in the presence of the sameelution medium used for in-vitro kinetic drug release analysis (e.g.PBS-Tween20, or mM Tris, 0.4 wt. % SDS, pH 7.4). Saturation of thesolution is prevented by frequent exchanges of the release medium withseveral volumes of fresh medium. TappingMode™ AFM imaging may be used toshow topography (a real-space projection of the coating surfacemicrostructure) and phase-angle changes of the AFM over the sample areato contrast differences in the materials properties. The AFM topographyimages can be three-dimensionally rendered to show the surface of acoated stent, which can show holes or voids of the coating which mayoccur as the polymer is absorbed and the drug is eluted over time, forexample.

Scanning Electron Microscopy (SEM) with Focused Ion Beam (FIB) MillingStents as described herein, and or produced by methods described hereinare visualized using SEM-FIB. Alternatively, a coated coupon could betested in this method. Focused ion beam FIB is a tool that allowsprecise site-specific sectioning, milling and depositing of materials.FIB can be used in conjunction with SEM, at ambient or cryo conditions,to produce in-situ sectioning followed by high-resolution imaging.FIB-SEM can produce a cross-sectional image of the polymer and druglayers on the stent. The image can be used to quantitate the thicknessof the layers and uniformity of the layer thickness at manufacture andat time points after stenting (or after in-vitro elution at various timepoints).

A FEI Dual Beam Strata 235 FIB/SEM system is a combination of a finelyfocused Ga ion beam (FIB) accelerated by 30 kV with a field emissionelectron beam in a scanning electron microscope instrument and is usedfor imaging and sectioning the stents. Both beams focus at the samepoint of the sample with a probe diameter less than 10 nm. The FIB canalso produce thinned down sections for TEM analysis.

To prevent damaging the surface of the stent with incident ions, a Ptcoating is first deposited via electron beam assisted deposition and ionbeam deposition prior to FIB sectioning. For FIB sectioning, the Ga ionbeam is accelerated to 30 kV and the sectioning process is about 2 h induration. Completion of the FIB sectioning allows one to observe andquantify by SEM the thickness of the polymer layers that are, forexample, left on the stent as they are absorbed.

Example 5 Analysis of the Thickness of a Device Coating

Analysis can be determined by either in-situ analysis or fromcross-sectioned samples.

X-Ray Photoelectron Spectroscopy (XPS)

XPS can be used to quantitatively determine the presence of elementalspecies and chemical bonding environments at the outer 5-10 nm of samplesurface. The technique can be operated in spectroscopy or imaging mode.When combined with a sputtering source XPS can be utilized to give depthprofiling chemical characterization. XPS (ESCA) and other analyticaltechniques such as described in Belu et al., “Three-DimensionalCompositional Analysis of Drug Eluting Stent Coatings Using ClusterSecondary Ion Mass Spectroscopy” Anal Chem. 80: 624-632 (2008)incorporated herein in its entirety by reference may be used.

Thus, in one test, a sample comprising a stent coated by methodsdescribed herein and/or a device as described herein is obtained. XPSanalysis is done on a sample using a Physical Electronics Quantum 2000Scanning ESCA. The monochromatic Al Kα source is operated at 15 kV witha power of 4.5 W. The analysis is done at a 45° take off angle. Threemeasurements are taken along the length of each sample with the analysisarea 20 microns in diameter. Low energy electron and Ar⁺ ion floods areused for charge compensation.

Time of Flight Secondary Ion Mass Spectrometery

TOF-SIMS can be used to determine molecular species (drug and polymer)at the outer 1-2 nm of sample surface when operated under staticconditions. The technique can be operated in spectroscopy or imagingmode at high spatial resolution. Additionally cross-sectioned samplescan be analysed. When operated under dynamic experimental conditions,known in the art, depth profiling chemical characterization can beachieved. For example, under static conditions (for example a ToF-SIMSIV (IonToF, Munster)) using a 25 Kv Bi⁺⁺ primary ion source maintainedbelow 1012 ions per cm² is used. Where necessary a low energy electronflood gun (0.6 nA DC) is used to charge compensate insulating samples.

Cluster Secondary Ion Mass Spectrometry, may be employed for depthprofiling as described Belu et al., “Three-Dimensional CompositionalAnalysis of Drug Eluting Stent Coatings Using Cluster Secondary Ion MassSpectroscopy” Anal. Chem. 80: 624-632 (2008) incorporated herein in itsentirety by reference.

A stent as described herein is obtained. The stent is prepared for SIMSanalysis by cutting it longitudinally and opening it up with tweezers.The stent is then pressed into multiple layers of iridium foil with theouter diameter facing outward.

TOF-SIMS experiments are performed on an Ion-TOF IV instrument equippedwith both Bi and SF5+ primary ion beam cluster sources. Sputter depthprofiling is performed in the dual-beam mode. The analysis source is apulsed, 25-keV bismuth cluster ion source, which bombarded the surfaceat an incident angle of 45° to the surface normal. The target current ismaintained at ˜0.3 pÅ (+10%) pulsed current with a raster size of 200um×200 um for all experiments. Both positive and negative secondary ionsare extracted from the sample into a reflectron-type time-of-flight massspectrometer. The secondary ions are then detected by a microchannelplate detector with a post-acceleration energy of 10 kV. A low-energyelectron flood gun is utilized for charge neutralization in the analysismode.

The sputter source used is a 5-keV SF5+ cluster source also operated atan incident angle of 45° to the surface normal. For thin model sampleson Si, the SF5+ current is maintained at ˜2.7 nÅ with a 750 um×750 umraster. For the thick samples on coupons and for the samples on stents,the current is maintained at 6 nA with a 500 um×500 um raster.

All primary beam currents are measured with a Faraday cup both prior toand after depth profiling.

All depth profiles are acquired in the noninterlaced mode with a 5-mspause between sputtering and analysis. Each spectrum is averaged over a7.37 second time period. The analysis is immediately followed by 15seconds of SF₅+ sputtering. For depth profiles of the surface andsubsurface regions only, the sputtering time was decreased to 1 secondfor the 5% active agent sample and 2 seconds for both the 25% and 50%active agent samples. Temperature-controlled depth profiles are obtainedusing a variable-temperature stage with Eurotherm Controls temperaturecontroller and IPSG V3.08 software. samples are first placed into theanalysis chamber at room temperature. The samples are brought to thedesired temperature under ultra high-vacuum conditions and are allowedto stabilize for 1 minute prior to analysis. All depth profilingexperiments are performed at −100 C and 25 C.

Atomic Force Microscopy (AFM)

AFM is a high resolution surface characterization technique. AFM is usedin the art to provide topographical imaging, in addition when employedin Tapping Mode™ can image material and or chemical properties of thesurface. Additionally cross-sectioned samples can be analyzed.

A stent as described herein is obtained. AFM may be alternatively beemployed as described in Ranade et al., “Physical characterization ofcontrolled release of paclitaxel from the TAXUS Express2 drug-elutingstent” J. Biomed. Mater. Res. 71(4):625-634 (2004) incorporated hereinin its entirety by reference.

Polymer and drug morphologies, coating composition, and cross-sectionalthickness at least may be determined using atomic force microscopy (AFM)analysis. A multi-mode AFM (Digital Instruments/Veeco Metrology, SantaBarbara, Calif.) controlled with Nanoscope IIIa and NanoScope Extenderelectronics is used TappingMode™ AFM imaging may be used to showtopography (a real-space projection of the coating surfacemicrostructure) and phase-angle changes of the AFM over the sample areato contrast differences in the materials properties. The AFM topographyimages can be three-dimensionally rendered to show the surface of acoated stent or cross-section. Scanning Electron Microscopy (SEM) withFocused Ion Beam (FIB) Stents as described herein, and or produced bymethods described herein are visualized using SEM-FIB analysis.Alternatively, a coated coupon could be tested in this method. Focusedion beam FIB is a tool that allows precise site-specific sectioning,milling and depositing of materials. FIB can be used in conjunction withSEM, at ambient or cryo conditions, to produce in-situ sectioningfollowed by high-resolution imaging. FIB-SEM can produce across-sectional image of the polymer layers on the stent. The image canbe used to quantitate the thickness of the layers as well as showwhether there is uniformity of the layer thickness at manufacture and attime points after stenting (or after in-vitro elution at various timepoints).

A FEI Dual Beam Strata 235 FIB/SEM system is a combination of a finelyfocused Ga ion beam (FIB) accelerated by 30 kV with a field emissionelectron beam in a scanning electron microscope instrument and is usedfor imaging and sectioning the stents. Both beams focus at the samepoint of the sample with a probe diameter less than 10 nm. The FIB canalso produce thinned down sections for TEM analysis.

To prevent damaging the surface of the stent with incident ions, a Ptcoating is first deposited via electron beam assisted deposition and ionbeam deposition prior to FIB sectioning. For FIB sectioning, the Ga ionbeam is accelerated to 30 kV and the sectioning process is about 2 h induration. Completion of the FIB sectioning allows one to observe andquantify by SEM the thickness of the polymer layers that are, forexample, left on the stent as they are absorbed.

Interferometry

Interferometry may additionally and/or alternatively used to determinethe thickness of the coating as noted in Belu et al., “Three-DimensionalCompositional Analysis of Drug Eluting Stent Coatings Using ClusterSecondary Ion Mass Spectroscopy” Anal. Chem. 80: 624-632 (2008)incorporated herein in its entirety by reference may be used.

Ellipsometry

Ellipsometry is sensitive measurement technique for coating analysis ona coupon. It uses polarized light to probe the dielectric properties ofa sample. Through an analysis of the state of polarization of the lightthat is reflected from the sample the technique allows the accuratecharacterization of the layer thickness and uniformity. Thicknessdeterminations ranging from a few angstroms to tens of microns arepossible for single layers or multilayer systems. See, for example,Jewell, et al., “Release of Plasmid DNA from Intravascular Stents Coatedwith Ultrathin Mulyikayered Polyelectrolyte Films” Biomacromolecules. 7:2483-2491 (2006) incorporated herein in its entirety by reference.

Example 6 Analysis of the Thickness of a Device Scanning ElectronMicroscopy (SEM)

A sample coated stent described herein is obtained. Thickness of thedevice can be assessed using this analytical technique. The thickness ofmultiple struts were taken to ensure reproducibility and to characterizethe coating and stent. The thickness of the coating was observed by SEMusing a Hitachi S-4800 with an accelerating voltage of 800V. Variousmagnifications are used. SEM can provide top-down and cross-sectionimages at various magnifications.

Nano X-Ray Computer Tomography

Another technique that may be used to view the physical structure of adevice in 3-D is Nano X-Ray Computer Tomography (e.g. such as made bySkyScan).

Example 7 Determination of the Type or Composition of a Polymer Coatinga Device Nuclear Magnetic Resonance (NMR)

Composition of the polymer samples before and after elution can bedetermined by ¹H NMR spectrometry as described in Xu et al.,“Biodegradation of poly(1-lactide-co-glycolide tube stents in bile”Polymer Degradation and Stability. 93:811-817 (2008) incorporated hereinin its entirety by reference. Compositions of polymer samples aredetermined for example using a 300M Bruker spectrometer withd-chloroform as solvent at room temperature.

Raman Spectroscopy

FT-Raman or confocal raman microscopy can be employed to determinecomposition.

For example, a sample (a coated stent) is prepared as described herein.Images are taken on the coating using Raman Spectroscopy. Alternatively,a coated coupon could be tested in this method. To test a sample usingRaman microscopy and in particular confocal Raman microscopy, it isunderstood that to get appropriate Raman high resolution spectrasufficient acquisition time, laser power, laser wavelength, sample stepsize and microscope objective need to be optimized. Raman spectroscopyand other analytical techniques such as described in Balss, et al.,“Quantitative spatial distribution of sirolimus and polymers indrug-eluting stents using confocal Raman microscopy” J. of BiomedicalMaterials Research Part A, 258-270 (2007), incorporated in its entiretyherein by reference, and/or described in Belu et al., “Three-DimensionalCompositional Analysis of Drug Eluting Stent Coatings Using ClusterSecondary Ion Mass Spectroscopy” Anal. Chem. 80: 624-632 (2008)incorporated herein in its entirety by reference may be used.

For example a WITec CRM 200 scanning confocal Raman microscope using aNd:YAG laser at 532 nm is applied in the Raman imaging mode. The sampleis placed upon a piezoelectrically driven table, the laser light isfocused upon the sample using a 10× dry objective (numerical aperture0.90), and the finely focused laser spot is scanned into the sample. Asthe laser scans the sample, over each 0.33 micron interval a Ramanspectrum with high signal to noise is collected using 0.3 Seconds ofintegration time. Each confocal cross-sectional image of the coatingsdisplays a region 70 μm wide by 10 μm deep, and results from thegathering of 6300 spectra with a total imaging time of 32 min.Multivariate analysis using reference spectra from samples of rapamycin(amorphous and crystalline) and polymer references are used todeconvolve the spectral data sets, to provide chemical maps of thedistribution.

In another test, spectral depth profiles of samples are performed with aCRM200 microscope system from WITec Instruments Corporation (Savoy,Ill.). The instrument is equipped with a NdYAG frequency doubled laser(532 excitation), a single monochromator (Acton) employing a 600groove/mm grating and a thermoelectrically cooled 1024 by 128 pixelarray CCD camera (Andor Technology). The microscope is equipped withappropriate collection optics that include a holographic laser bandpassrejection filter (Kaiser Optical Systems Inc.) to minimize Rayleighscatter into the monochromator. The Raman scattered light are collectedwith a 50 micron optical fiber. Using the “Raman Spectral Imaging” modeof the instrument, spectral images are obtained by scanning the samplein the x, z direction with a piezo driven xyz scan stage and collectinga spectrum at every pixel. Typical integration times are 0.3 s perpixel. The spectral images are 4800 total spectra corresponding to aphysical scan dimension of 40 by 20 microns. For presentation of theconfocal Raman data, images are generated base don unique properties ofthe spectra (i.e. integration of a Raman band, band height intensity, orband width). The microscope stage is modified with a custom-built sampleholder that positioned and rotated the stents around their primary axis.The x direction is defined as the direction running parallel to thelength of the stent and the z direction refers to the directionpenetrating through the coating from the air-coating to thecoating-metal interface. Typical laser power is <10 mW on the samplestage. All experiments can be conducted with a plan achromat objective,100×N_(A)=0.9 (Nikon).

Samples (n=5) comprising stents made of L605 and having coatings asdescribed herein and/or produced by methods described herein can beanalyzed. For each sample, three locations are selected along the stentlength. The three locations are located within one-third portions of thestents so that the entire length of the stent are represented in thedata. The stent is then rotated 180 degrees around the circumference andan additional three locations are sampled along the length. In eachcase, the data is collected from the strut portion of the stetn. Sixrandom spatial locations are also profiled on coated coupon samples madeof L605 and having coatings as described herein and/or produced bymethods described herein. The Raman spectra of each individual componentpresent in the coatings are also collected for comparison and reference.Using the instrument software, the average spectra from the spectralimage data are calculated by selecting the spectral image pixels thatare exclusive to each layer. The average spectra are then exported intoGRAMS/AI v. 7.02 software (Thermo Galactic) and the appropriate Ramanbands are fit to a Voigt function. The band areas and shift positionsare recorded.

The pure component spectrum for each component of the coating (e.g.drug, polymer) are also collected at 532 and 785 nm excitation. The 785nm excitation spectra are collected with a confocal Raman microscope(WITec Instruments Corp. Savoy, Ill.) equipped with a 785 nm diodelaser, appropriate collection optics, and a back-illuminatedthermoelectriaclly cooled 1024×128 pixel array CCD camera optimized forvisible and infrared wavelengths (Andor Technology).

Time of Flight Secondary Ion Mass Spectrometery

TOF-SIMS can be used to determine molecular species (drug and polymer)at the outer 1-2 nm of sample surface when operated under staticconditions. The technique can be operated in spectroscopy or imagingmode at high spatial resolution. Additionally cross-sectioned samplescan be analysed. When operated under dynamic experimental conditions,known in the art, depth profiling chemical characterization can beachieved.

For example, under static conditions (for example a ToF-SIMS IV (IonToF,Munster)) using a 25 Kv Bi⁺⁺ primary ion source maintained below 10¹²ions per cm² is used. Where necessary a low energy electron flood gun(0.6 nA DC) is used to charge compensate insulating samples.

Cluster Secondary Ion Mass Spectrometry, may be employed as describedBelu et al, “Three-Dimensional Compositional Analysis of Drug ElutingStent Coatings Using Cluster Secondary Ion Mass Spectroscopy” Anal.Chem. 80: 624-632 (2008) incorporated herein in its entirety byreference.

A stent as described herein is obtained. The stent is prepared for SIMSanalysis by cutting it longitudinally and opening it up with tweezers.The stent is then pressed into multiple layers of iridium foil with theouter diameter facing outward.

TOF-SIMS experiments are performed on an Ion-TOF IV instrument equippedwith both Bi and SF5+ primary ion beam cluster sources. Sputter depthprofiling is performed in the dual-beam mode. The analysis source is apulsed, 25-keV bismuth cluster ion source, which bombarded the surfaceat an incident angle of 45° to the surface normal. The target current ismaintained at ˜0.3 pÅ (+10%) pulsed current with a raster size of 200um×200 um for all experiments. Both positive and negative secondary ionsare extracted from the sample into a reflectron-type time-of-flight massspectrometer. The secondary ions are then detected by a microchannelplate detector with a post-acceleration energy of 10 kV. A low-energyelectron flood gun is utilized for charge neutralization in the analysismode.

The sputter source used is a 5-keV SF5+ cluster source also operated atan incident angle of 45° to the surface normal. For thin model sampleson Si, the SF5+ current is maintained at ˜2.7 nÅ with a 750 um×750 umraster. For the thick samples on coupons and for the samples on stents,the current is maintained at 6 nA with a 500 um×500 um raster. Allprimary beam currents are measured with a Faraday cup both prior to andafter depth profiling.

All depth profiles are acquired in the noninterlaced mode with a 5-mspause between sputtering and analysis. Each spectrum is averaged over a7.37 second time period. The analysis is immediately followed by 15seconds of SF₅ ⁺ sputtering. For depth profiles of the surface andsubsurface regions only, the sputtering time was decreased to 1 secondfor the 5% active agent sample and 2 seconds for both the 25% and 50%active agent samples. Temperature-controlled depth profiles are obtainedusing a variable-temperature stage with Eurotherm Controls temperaturecontroller and IPSG V3.08 software. samples are first placed into theanalysis chamber at room temperature. The samples are brought to thedesired temperature under ultra high-vacuum conditions and are allowedto stabilize for 1 minute prior to analysis. All depth profilingexperiments are performed at −100 C and 25 C.

Atomic Force Microscopy (AFM)

AFM is a high resolution surface characterization technique. AFM is usedin the art to provide topographical imaging, in addition when employedin Tapping Mode™ can image material and or chemical properties of thesurface. Additionally cross-sectioned samples can be analyzed. Coatingcomposition may be determined using Tapping Mode™ atomic forcemicroscopy (AFM) analysis. Other modes of operation are well known andcan be employed here by those skilled in the art.

A stent as described herein is obtained. AFM may be employed asdescribed in Ranade et al., “Physical characterization of controlledrelease of paclitaxel from the TAXUS Express2 drug-eluting stent” J.Biomed. Mater. Res. 71(4):625-634 (2004) incorporated herein in itsentirety by reference.

Polymer and drug morphologies, coating composition, at least may bedetermined using atomic force microscopy (AFM) analysis. A multi-modeAFM (Digital Instruments/Veeco Metrology, Santa Barbara, Calif.)controlled with Nanoscope IIIa and NanoScope Extender electronics isused. TappingMode™ AFM imaging may be used to show topography (areal-space projection of the coating surface microstructure) andphase-angle changes of the AFM over the sample area to contrastdifferences in the materials properties.

Infrared (IR) Spectroscopy for In-Vitro Testing

Infrared (IR) Spectroscopy using FTIR, ATR-IR or micro ATR-IR can beused to identify polymer composition by comparison to standard polymerreference spectra.

Example 8 Determination of the Bioabsorbability of a Device

In some embodiments of the device the substrate coated itself is made ofa bioabsorbable material, such as the bioabsorbable polymers presentedherein, or another bioabsorbable material such as magnesium and, thus,the entire device is bioabsorbable. Techniques presented with respect toshowing Bioabsorbability of a polymer coating may be used toadditionally and/or alternatively show the bioabsorbability of a device,for example, by GPC In-Vivo testing, HPLC In-Vivo Testing, GPC In-Vitrotesting, HPLC In-Vitro Testing, SEM-FIB Testing, Raman Spectroscopy,SEM, and XPS as described herein with variations and adjustments whichwould be obvious to those skilled in the art. Another technique to viewthe physical structure of a device in 3-D is Nano X-Ray ComputerTomography (e.g. such as made by SkyScan), which could be used in anelution test and/or bioabsorbability test, as described herein to showthe physical structure of the coating remaining on stents at each timepoint, as compared to a scan prior to elution/bioabsorbtion.

Example 9 Determination of Secondary Structures Presence of a BiologicalAgent Raman Spectroscopy

FT-Raman or confocal raman microscopy can be employed to determinesecondary structure of a biological Agent. For example fitting of theAmide I, II, or III regions of the Raman spectrum can elucidatesecondary structures (e.g. alpha-helices, beta-sheets). See, forexample, Iconomidou, et al., “Secondary Structure of Chorion Proteins ofthe Teleosetan Fish Dentex dentex by ATR FR-IR and FT-RamanSpectroscopy” J. of Structural Biology, 132, 112-122 (2000); Griebenow,et al., “On Protein Denaturation in Aqueous-Organic Mixtures but Not inPure Organic Solvents” J. Am. Chem. Soc., Vol 118, No. 47, 11695-11700(1996).

Infrared (IR) Spectroscopy for In-Vitro Testing

Infrared spectroscopy, for example FTIR, ATR-IR and micro ATR-IR can beemployed to determine secondary structure of a biological Agent. Forexample fitting of the Amide I, II, of III regions of the infraredspectrum can elucidate secondary structures (e.g. alpha-helices,beta-sheets).

Example 10 Determination of the Microstructure of a Coating on a MedicalDevice Atomic Force Microscopy (AFM)

AFM is a high resolution surface characterization technique. AFM is usedin the art to provide topographical imaging, in addition when employedin Tapping Mode™ can image material and or chemical properties of thesurface. Additionally cross-sectioned samples can be analyzed. Thetechnique can be used under ambient, solution, humidified or temperaturecontrolled conditions. Other modes of operation are well known and canbe readily employed here by those skilled in the art.

A stent as described herein is obtained. AFM is used to determine themicrostructure of the coating. A stent as described herein is obtained.AFM may be employed as described in Ranade et al., “Physicalcharacterization of controlled release of paclitaxel from the TAXUSExpress2 drug-eluting stent” J. Biomed. Mater. Res. 71(4):625-634 (2004)incorporated herein in its entirety by reference.

For example, polymer and drug morphologies, coating composition, andphysical structure may be determined using atomic force microscopy (AFM)analysis. A multi-mode AFM (Digital Instruments/Veeco Metrology, SantaBarbara, Calif.) controlled with Nanoscope IIIa and NanoScope Extenderelectronics is used. Samples are examined in the dry state using AFMbefore elution of the drug (e.g. rapamycin). Samples are also examinedat select time points through a elution period (e.g. 48 hours) by usingan AFM probe-tip and flow-through stage built to permit analysis of wetsamples. The wet samples are examined in the presence of the sameelution medium used for in-vitro kinetic drug release analysis (e.g.PBS-Tween20, or 10 mM Tris, 0.4 wt. % SDS, pH 7.4). Saturation of thesolution is prevented by frequent exchanges of the release medium withsever volumes of fresh medium. TappingMode™ AFM imaging may be used toshow topography (a real-space projection of the coating surfacemicrostructure) and phase-angle changes of the AFM over the sample areato contrast differences in the materials properties. The AFM topographyimages can be three-dimensionally rendered to show the surface of acoated stent, which can show holes or voids of the coating which mayoccur as the polymer is absorbed and the drug is released from thepolymer over time, for example.

Nano X-Ray Computer Tomography

Another technique that may be used to view the physical structure of adevice in 3-D is Nano X-Ray Computer Tomography (e.g. such as made bySkyScan), which could be used in an elution test and/or bioabsorbabilitytest, as described herein to show the physical structure of the coatingremaining on stents at each time point, as compared to a scan prior toelution/bioabsorbtion.

Example 11 Determination of an Elution Profile In Vitro Example 11a

In one method, a stent described herein is obtained. The elution profileis determined as follows: stents are placed in 16 mL test tubes and 15mL of 10 mM PBS (pH 7.4) is pipetted on top. The tubes are capped andincubated at 37 C with end-over-end rotation at 8 rpm. Solutions arethen collected at the designated time points (e.g. 1d, 7d, 14d, 21d, and28d) (e.g. 1 week, 2 weeks, and 10 weeks) and replenished with fresh 1.5ml solutions at each time point to prevent saturation. One mL of DCM isadded to the collected sample of buffer and the tubes are capped andshaken for one minute and then centrifuged at 200×G for 2 minutes. Thesupernatant is discarded and the DCM phase is evaporated to drynessunder gentle heat (40° C.) and nitrogen gas. The dried DCM isreconstituted in 1 mL of 60:40 acetonitrile:water (v/v) and analyzed byHPLC. HPLC analysis is performed using Waters HPLC system (mobile phase58:37:5 acetonitrile:water:methanol 1 mL/min, 20 uL injection, C18Novapak Waters column with detection at 232 nm).

Example 11b

In another method, the in vitro pharmaceutical agent elution profile isdetermined by a procedure comprising contacting the device with anelution media comprising ethanol (5%) wherein the pH of the media isabout 7.4 and wherein the device is contacted with the elution media ata temperature of about 37° C. The elution media containing the device isoptionally agitating the elution media during the contacting step. Thedevice is removed (and/or the elution media is removed) at least atdesignated time points (e.g. 1 h, 3 h, 5 h, 7 h, 1d, and daily up to28d) (e.g. 1 week, 2 weeks, and 10 weeks). The elution media is thenassayed using a UV-Vis for determination of the pharmaceutical agentcontent. The elution media is replaced at each time point with freshelution media to avoid saturation of the elution media. Calibrationstandards containing known amounts of drug were also held in elutionmedia for the same durations as the samples and used at each time pointto determine the amount of drug eluted at that time (in absolute amountand as a cumulative amount eluted).

In one test, devices were coated tested using this method. In theseexperiments two different polymers were employed: Polymer A: −50:50PLGA-Ester End Group, MW ˜19 kD, degradation rate 70 days; Polymer B:˜50:50 PLGA-Carboxylate End Group, MW˜10 kD, degradation rate 28 days.Metal stents were coated as follows: AS1: (n=6) PolymerA/Rapamycin/Polymer A/Rapamycin/Polymer A; AS2: (n=6) PolymerA/Rapamycin/Polymer A/Rapamycin/Polymer B; AS1 (213): (n=6) PolymerB/Rapamycin/Polymer B/Rapamycin/Polymer B; AS1b: (n=6) PolymerA/Rapamycin/Polymer A/Rapamycin/Polymer A; AS2b: (n=6) PolymerA/Rapamycin/Polymer A/Rapamycin/Polymer B. The in vitro pharmaceuticalagent elution profile was determined by contacting each device with anelution media comprising ethanol (5%) wherein the pH of the media isabout 7.4 and wherein the device was contacted with the elution media ata temperature of about 37° C. The elution media was removed from devicecontact at least at 1 h, 3 h, 5 h, 7 h, 1d, and at additional timepoints up to 70 days (See FIGS. 5-8). The elution media was then assayedusing a UV-Vis for determination of the pharmaceutical agent content (inabsolute amount and cumulative amount eluted). The elution media wasreplaced at each time point with fresh elution media to avoid saturationof the elution media. Calibration standards containing known amounts ofdrug were also held in elution media for the same durations as thesamples and assayed by UV-Vis at each time point to determine the amountof drug eluted at that time (in absolute amount and as a cumulativeamount eluted), compared to a blank comprising Spectroscopic gradeethanol. Elution profiles as shown in FIGS. 5-8, showing the averageamount of rapamycin eluted at each time point (average of all stentstested) in micrograms. Table 2 shows for each set of stents (n=6) ineach group (AS1, AS2, AS(213), AS1b, AS2b), the average amount ofrapamycin in ug loaded on the stents, the average amount of polymer inug loaded on the stents, and the total amount of rapamycin and polymerin ug loaded on the stents.

TABLE 2 Ave. Stent Ave. Ave. Total Coating Rapa, ug Poly, ug Mass, ugAS1 175 603 778 AS2 153 717 870 AS1(213) 224 737 961 AS1b 171 322 493AS2b 167 380 547

Example 11c

In another method, the in vitro pharmaceutical agent elution profile isdetermined by a procedure comprising contacting the device with anelution media comprising ethanol (20%) and phosphate buffered saline(80%) wherein the pH of the media is about 7.4 and wherein the device iscontacted with the elution media at a temperature of about 37° C. Theelution media containing the device is optionally agitating the elutionmedia during the contacting step. The device is removed (and/or theelution media is removed) at least at designated time points (e.g. 1 h,3 h, 5 h, 7 h, 1d, and daily up to 28d) (e.g. 1 week, 2 weeks, and 10weeks). The elution media is replaced periodically (at least at eachtime point, and/or daily between later time points) to preventsaturation; the collected media are pooled together for each time point.The elution media is then assayed for determination of thepharmaceutical agent content using HPLC. The elution media is replacedat each time point with fresh elution media to avoid saturation of theelution media. Calibration standards containing known amounts of drugare also held in elution media for the same durations as the samples andused at each time point to determine the amount of drug eluted at thattime (in absolute amount and as a cumulative amount eluted). Where theelution method changes the drug over time, resulting in multiple peakspresent for the drug when tested, the use of these calibration standardswill also show this change, and allows for adding all the peaks to givethe amount of drug eluted at that time period (in absolute amount and asa cumulative amount eluted).

In one test, devices (n=9, laminate coated stents) as described hereinwere coated and tested using this method. In these experiments a singlepolymer was employed: Polymer A: 50:50 PLGA-Ester End Group, MW˜19 kD.The metal (stainless steel) stents were coated as follows: PolymerA/Rapamycin/Polymer A/Rapamycin/Polymer A, and the average amount ofrapamycin on each stent was 162 ug (stdev 27 ug). The coated stents werecontacted with an elution media (5.00 mL) comprising ethanol (20%) andphosphate buffered saline wherein the pH of the media is about 7.4(adjusted with potassiume carbonate solution—1 g/100 mL distilled water)and wherein the device is contacted with the elution media at atemperature of about 37° C.+/−0.2° C. The elution media containing thedevice was agitated in the elution media during the contacting step. Theelution media was removed at least at time points of 1 h, 3 h, 5 h, 7 h,1d, and daily up to 28d. The elution media was assayed for determinationof the pharmaceutical agent (rapamycin) content using HPLC. The elutionmedia was replaced at each time point with fresh elution media to avoidsaturation of the elution media. Calibration standards containing knownamounts of drug were also held in elution media for the same durationsas the samples and assayed at each time point to determine the amount ofdrug eluted at that time (in absolute amount and as a cumulative amounteluted). The multiple peaks present for the rapamycin (also present inthe calibration standards) were added to give the amount of drug elutedat that time period (in absolute amount and as a cumulative amounteluted). HPLC analysis is performed using Waters HPLC system, set up andrun on each sample as provided in the Table 3 below using an injectionvolume of 100 uL.

TABLE 3 Time point % Ammonium Acetate Flow Rate (minutes) % Acetonitrile(0.5%), pH 7.4 (mL/min) 0.00 10 90 1.2 1.00 10 90 1.2 12.5 95 5 1.2 13.5100 0 1.2 14.0 100 0 3 16.0 100 0 3 17.0 10 90 2 20.0 10 90 0

FIG. 9 elution profiles resulted, showing the average cumulative amountof rapamycin eluted at each time point (average of n=9 stents tested) inmicrograms. FIG. 10 also expresses the same elution profile, graphed ona logarithmic scale (x-axis is log(time)).

Example 11d

To obtain an accelerated in-vitro elution profile, an acceleratedelution buffer comprising 18% v/v of a stock solution of 0.067 mol/LKH2PO4 and 82% v/v of a stock solution of 0.067 mol/L Na2HPO4 with a pHof 7.4 is used. Stents described herein are expanded and then placed in1.5 ml solution of this accelerated elution in a 70° C. bath withrotation at 70 rpm. The solutions are then collected at the followingtime points: 0 min., 15 min., 30 min., 1 hr, 2 hr, 4 hr, 6 hr, 8 hr, 12hr, 16 hr, 20 hr, 24 hr, 30 hr, 36 hr and 48 hr. Fresh acceleratedelution buffer are added periodically at least at each time point toreplace the incubated buffers that are collected and saved in order toprevent saturation. For time points where multiple elution media areused (refreshed between time points), the multiple collected solutionsare pooled together for liquid extraction by dichloromethane.Dichloromethane extraction and HPLC analysis is performed in the mannerdescribed previously.

In Vivo Example 11e

Rabbit in vivo models as described above are euthanized at multiple timepoints. Stents are explanted from the rabbits. The explanted stents areplaced in 16 mL test tubes and 15 mL of 10 mM PBS (pH 7.4) is pipette ontop. One mL of DCM is added to the buffer and the tubes are capped andshaken for one minute and then centrifuged at 200×G for 2 minutes. Thesupernatant is discarded and the DCM phase is evaporated to drynessunder gentle heat (40° C.) and nitrogen gas. The dried DCM isreconstituted in 1 mL of 60:40 acetonitrile:water (v/v) and analyzed byHPLC. HPLC analysis is performed using Waters HPLC system (mobile phase58:37:5 acetonitrile:water:methanol 1 mL/min, 20 uL injection, C18Novapak Waters column with detection at 232 nm).

Example 12 Determination of the Conformability (Conformality) of aDevice Coating

The ability to uniformly coat arterial stents with controlledcomposition and thickness using electrostatic capture in a rapidexpansion of supercritical solution (RESS) experimental series has beendemonstrated.

Scanning Electron Microscopy (SEM)

Stents are observed by SEM using a Hitachi S-4800 with an acceleratingvoltage of 800V. Various magnifications are used to evaluate theintegrity, especially at high strain regions. SEM can provide top-downand cross-section images at various magnifications. Coating uniformityand thickness can also be assessed using this analytical technique. Pre-and post-expansions stents are observed by SEM using a Hitachi S-4800with an accelerating voltage of 800V. Various magnifications are used toevaluate the integrity of the layers, especially at high strain regions.

Scanning Electron Microscopy (SEM) with Focused Ion Beam (FIB) Stents asdescribed herein, and or produced by methods described herein arevisualized using SEM-FIB analysis. Alternatively, a coated coupon couldbe tested in this method. Focused ion beam FIB is a tool that allowsprecise site-specific sectioning, milling and depositing of materials.FIB can be used in conjunction with SEM, at ambient or cryo conditions,to produce in-situ sectioning followed by high-resolution imaging.Cross-sectional FIB images may be acquired, for example, at 7000× and/orat 20000× magnification. An even coating of consistent thickness isvisible.

Optical Microscopy

An Optical microscope may be used to create and inspect the stents andto empirically survey the coating of the substrate (e.g. coatinguniformity). Nanoparticles of the drug and/or the polymer can be seen onthe surfaces of the substrate using this analytical method. Followingsintering, the coatings can be see using this method to view the coatingconformably and for evidence of crystallinity of the drug.

Example 13 Determination of the Total Content of the Active Agent

Determination of the total content of the active agent in a coated stentmay be tested using techniques described herein as well as othertechniques obvious to one of skill in the art, for example using GPC andHPLC techniques to extract the drug from the coated stent and determinethe total content of drug in the sample.

UV-VIS can be used to quantitatively determine the mass of rapamycincoated onto the stents. A UV-Vis spectrum of Rapamycin can be shown anda Rapamycin calibration curve can be obtained, (e.g. λ@277 nm inethanol). Rapamycin is then dissolved from the coated stent in ethanol,and the drug concentration and mass calculated.

In one test, the total amount of rapamycin present in units ofmicrograms per stent is determined by reverse phase high performanceliquid chromatography with UV detection (RP-HPLC-UV). The analysis isperformed with modifications of literature-based HPLC methods forrapamycin that would be obvious to a person of skill in the art. Theaverage drug content of samples (n=10) from devices comprising stentsand coatings as described herein, and/or methods described herein aretested.

Example 14 Determination of the Extent of Aggregation of an Active AgentRaman Spectroscopy

Confocal Raman microscopy can be used to characterize the drugaggregation by mapping in the x-y or x-z direction. Additionallycross-sectioned samples can be analysed. Raman spectroscopy and otheranalytical techniques such as described in Balss, et al., “Quantitativespatial distribution of sirolimus and polymers in drug-eluting stentsusing confocal Raman microscopy” J. of Biomedical Materials ResearchPart A, 258-270 (2007), incorporated in its entirety herein byreference, and/or described in Belu et al., “Three-DimensionalCompositional Analysis of Drug Eluting Stent Coatings Using ClusterSecondary Ion Mass Spectroscopy” Anal. Chem. 80: 624-632 (2008)incorporated herein in its entirety by reference may be used.

A sample (a coated stent) is prepared as described herein. Images aretaken on the coating using Raman Spectroscopy. Alternatively, a coatedcoupon could be tested in this method. A WITec CRM 200 scanning confocalRaman microscope using a NiYAG laser at 532 nm is applied in the Ramanimaging mode. The sample is place upon a piezoelectrically driven table,the laser light is focused upon the sample using a 100× dry objective(numerical aperture 0.90), and the finely focused laser spot is scannedinto the sample. As the laser scans the sample, over each 0.33 microninterval a Raman spectrum with high signal to noise is collected using0.3 Seconds of integration time. Each confocal cross-sectional image ofthe coatings displays a region 70 μm wide by 10 μm deep, and resultsfrom the gathering of 6300 spectra with a total imaging time of 32 min.To deconvolute the spectra and obtain separate images of the activeagent and the polymer, all the spectral data (6300 spectra over theentire spectral region 500-3500 cm−1) are processed using an augmentedclassical least squares algorithm (Eigenvector Research, WenatcheeWash.) using basis spectra obtained from samples of rapamycin (amorphousand crystalline) and polymer. For each sample, several areas aremeasured by Raman to ensure that results are reproducible, and to showlayering of drug and polymer through the coating. Confocal RamanSpectroscopy can profile down micron by micron, can show the compositionof the coating through the thickness of the coating.

Time of Flight Secondary Ion Mass Spectrometery

TOF-SIMS can be used to determine drug aggregation at the outer 1-2 nmof sample surface when operated under static conditions. The techniquecan be operated in spectroscopy or imaging mode at high spatialresolution. Additionally cross-sectioned samples can be analysed. Whenoperated under dynamic experimental conditions, known in the art, depthprofiling chemical characterization can be achieved.

For example, under static conditions (for example a ToF-SIMS IV (IonToF,Munster)) using a 25 Kv Bi⁺⁺ primary ion source maintained below 10¹²ions per cm² is used. Where necessary a low energy electron flood gun(0.6 nA DC) is used to charge compensate insulating samples.

Cluster Secondary Ion Mass Spectrometry, may be employed as described inBelu et al., “Three-Dimensional Compositional Analysis of Drug ElutingStent Coatings Using Cluster Secondary Ion Mass Spectroscopy” Anal.Chem. 80: 624-632 (2008) incorporated herein in its entirety byreference.

A stent as described herein is obtained. The stent is prepared for SIMSanalysis by cutting it longitudinally and opening it up with tweezers.The stent is then pressed into multiple layers of iridium foil with theouter diameter facing outward.

For example TOF-SIMS experiments are performed on an Ion-TOF IVinstrument equipped with both Bi and SF5+ primary ion beam clustersources. Sputter depth profiling is performed in the dual-beam mode. Theanalysis source is a pulsed, 25-keV bismuth cluster ion source, whichbombarded the surface at an incident angle of 45° to the surface normal.The target current is maintained at ˜0.3 pÅ (+10%) pulsed current with araster size of 200 um×200 um for all experiments. Both positive andnegative secondary ions are extracted from the sample into areflectron-type time-of-flight mass spectrometer. The secondary ions arethen detected by a microchannel plate detector with a post-accelerationenergy of 10 kV. A low-energy electron flood gun is utilized for chargeneutralization in the analysis mode.

The sputter source used is a 5-keV SF5+ cluster source also operated atan incident angle of 45° to the surface normal. For thin model sampleson Si, the SF5+ current is maintained at ˜2.7 nA with a 750 um×750 umraster. For the thick samples on coupons and for the samples on stents,the current is maintained at 6 nA with a 500 um×500 um raster. Allprimary beam currents are measured with a Faraday cup both prior to andafter depth profiling.

All depth profiles are acquired in the noninterlaced mode with a 5-mspause between sputtering and analysis. Each spectrum is averaged over a7.37 second time period. The analysis is immediately followed by 15seconds of SF5+ sputtering. For depth profiles of the surface andsubsurface regions only, the sputtering time was decreased to 1 secondfor the 5% active agent sample and 2 seconds for both the 25% and 50%active agent samples.

Temperature-controlled depth profiles are obtained using avariable-temperature stage with Eurotherm Controls temperaturecontroller and IPSG V3.08 software. samples are first placed into theanalysis chamber at room temperature. The samples are brought to thedesired temperature under ultra high-vacuum conditions and are allowedto stabilize for 1 minute prior to analysis. All depth profilingexperiments are performed at −100 C and 25 C.

Atomic Force Microscopy (AFM)

AFM is a high resolution surface characterization technique. AFM is usedin the art to provide topographical imaging, in addition when employedin Tapping Mode™ can image material and or chemical properties forexample imaging drug in an aggregated state. Additionallycross-sectioned samples can be analyzed.

A stent as described herein is obtained. AFM may be employed asdescribed in Ranade et al., “Physical characterization of controlledrelease of paclitaxel from the TAXUS Express2 drug-eluting stent” J.Biomed. Mater. Res. 71(4):625-634 (2004) incorporated herein in itsentirety by reference.

Polymer and drug morphologies, coating composition, at least may bedetermined using atomic force microscopy (AFM) analysis. A multi-modeAFM (Digital Instruments/Veeco Metrology, Santa Barbara, Calif.)controlled with Nanoscope IIIa and NanoScope Extender electronics isused. TappingMode™ AFM imaging may be used to show topography (areal-space projection of the coating surface microstructure) andphase-angle changes of the AFM over the sample area to contrastdifferences in the materials properties.

Example 15 Determination of the Blood Concentration of an Active Agent

This assay can be used to demonstrate the relative efficacy of atherapeutic compound delivered from a device of the invention to notenter the blood stream and may be used in conjunction with a drugpenetration assay (such as is described in PCT/US2006/010700,incorporated in its entirety herein by reference). At predetermined timepoints (e.g. 1d, 7d, 14d, 21d, and 28d, or e.g. 6 hrs, 12 hrs, 24 hrs,36 hrs, 2d, 3d, 5d, 7d, 8d, 14d, 28d, 30d, and 60d), blood samples fromthe subjects that have devices that have been implanted are collected byany art-accepted method, including venipuncture. Blood concentrations ofthe loaded therapeutic compounds are determined using any art-acceptedmethod of detection, including immunoassay, chromatography (includingliquid/liquid extraction HPLC tandem mass spectrometric method(LC-MS/MS), and activity assays. See, for example, Ji, et al., “96-Wellliquid-liquid extraction liquid chromatography-tandem mass spectrometrymethod for the quantitative determination of ABT-578 in human bloodsamples” Journal of Chromatography B. 805:67-75 (2004) incorporated inits entirety herein by reference.

In one test, blood samples are collected by venipuncture into evacuatedcollection tubes containing editic acid (EDTA) (n=4). Bloodconcentrations of the active agent (e.g. rapamycin) are determined usinga validated liquid/liquid extraction HPLC tandem pass mass spectormetricmethod (LC-MS/MS) (Ji et al., et al., 2004). The data are averaged, andplotted with time on the x-axis and blood concetration of the drug isrepresented on the y-axis in ng/ml.

Example 16 Preparation of Supercritical Solution ComprisingPoly(Lactic-Co-Glycolic Acid) (PLGA) in Hexafluoropropane

A view cell at room temperature (with no applied heat) is pressurizedwith filtered 1,1,1,2,3,3-Hexafluoropropane until it is full and thepressure reaches 4500 psi. Poly(lactic-co-glycolic acid) (PLGA) is addedto the cell for a final concentration of 2 mg/ml. The polymer is stirredto dissolve for one hour. The polymer is fully dissolved when thesolution is clear and there are no solids on the walls or windows of thecell.

Example 17 Dry Powder Rapamycin Coating on an Electrically Charged L605Cobalt Chromium Metal Coupon

A 1 cm×2 cm L605 cobalt chromium metal coupon serving as a targetsubstrate for rapamycin coating is placed in a vessel and attached to ahigh voltage electrode. Alternatively, the substrate may be a stent oranother biomedical device as described herein, for example. The vessel(V), of approximately 1500 cm³ volume, is equipped with two separatenozzles through which rapamycin or polymers could be selectivelyintroduced into the vessel. Both nozzles are grounded. Additionally, thevessel (V) is equipped with a separate port was available for purgingthe vessel. Upstream of one nozzle (D) is a small pressure vessel (PV)approximately 5 cm³ in volume with three ports to be used as inlets andoutlets. Each port is equipped with a valve which could be actuatedopened or closed. One port, port (1) used as an inlet, is an additionport for the dry powdered rapamycin. Port (2), also an inlet is used tofeed pressurized gas, liquid, or supercritical fluid into PV. Port (3),used as an outlet, is used to connect the pressure vessel (PV) withnozzle (D) contained in the primary vessel (V) with the target coupon.

Dry powdered Rapamycin obtained from LC Laboratories in a predominantlycrystalline solid state, 50 mg milled to an average particle size ofapproximately 3 microns, is loaded into (PV) through port (1) then port(1) is actuated to the closed position. The metal coupon is then chargedto +7.5 kV using a Glassman Series EL high-voltage power source. Thedrug nozzle on port has a voltage setting of −7.5 kV. Afterapproximately 60-seconds, the drug is injected and the voltage iseliminated. Upon visual inspection of the coupon using an opticalmicroscope, the entire surface area of the coupon is examined forrelatively even distribution of powdered material. X-ray diffraction(XRD) is performed as described herein to confirm that the powderedmaterial is largely crystalline in nature as deposited on the metalcoupon. UV-Vis and FTIR spectroscopy is performed as describe herein toconfirm that the material deposited on the coupon is rapamycin.

Example 18 Polymer Coating on an Electrically Charged L605 Coupon UsingRapid Expansion from a Liquefied Gas

A coating apparatus as described in example 17 above is used in theforegoing example. In this example the second nozzle, nozzle (P), isused to feed precipitated polymer particles into vessel (V) to coat aL605 coupon. Alternatively, the substrate may be a stent or anotherbiomedical device as described herein, for example. Nozzle (P) isequipped with a heater and controller to minimize heat loss due to theexpansion of liquefied gases. Upstream of nozzle (P) is a pressurevessel, (PV2), with approximately 25-cm³ internal volume. The pressurevessel (PV2) is equipped with multiple ports to be used for inlets,outlets, thermocouples, and pressure transducers. Additionally, (PV2) isequipped with a heater and a temperature controller. Each port isconnected to the appropriate valves, metering valves, pressureregulators, or plugs to ensure adequate control of material into and outof the pressure vessel (PV2). One outlet from (PV2) is connected to ametering valve through pressure rated tubing which was then connected tonozzle (P) located in vessel (V). In the experiment, 150 mg ofpoly(lactic-co-glycolic acid) (PLGA) is added to pressure vessel (PV2).1,1,1,2,3,3-hexafluoropropane is added to the pressure vessel (PV2)through a valve and inlet. Pressure vessel (PV2) is set at roomtemperature with no applied heat and the pressure is 4500 psi. Nozzle(P) is heated to 150° C. A 1-cm×2-cm L605 coupon is placed into vessel(V), attached to an electrical lead and heated via a heat block 110° C.Nozzle (P) is attached to ground. The voltage is set on the polymerspray nozzle and an emitter=pair beaker to a achieve a current greaterthan or equal to 0.02 mAmps using a Glassman high-voltage power sourceat which point the metering valve is opened between (PV2) and nozzle (P)in pressure vessel (PV). Polymer dissolved in liquefied gas and is fedat a constant pressure of 200 psig into vessel (V) maintained atatmospheric pressure through nozzle (P) at an approximate rate of 3.0cm³/min. After approximately 5 seconds, the metering valve is closeddiscontinuing the polymer-solvent feed. Vessel (V) is Nitrogen gas for30 seconds to displace the fluorocarbon. After approximately 30 seconds,the metering valve is again opened for a period of approximately 5seconds and then closed. This cycle is repeated about 4 times. After anadditional 1-minute the applied voltage to the coupon was discontinuedand the coupon was removed from pressure vessel (V). Upon inspection byoptical microscope, a polymer coating is examined for even distributionon all non-masked surfaces of the coupon.

Example 19 Dual Coating of a Metal Coupon with Crystalline Rapamycin andpoly(lactic-co-glycolic acid) (PLGA)

An apparatus described in example 17 and further described in example 18is used in the foregoing example. In preparation for the coatingexperiment, 25 mg of crystalline powdered rapamycin with an averageparticle size of 3-microns is added to (PV) through port (1), then port(1) was closed. Next, 150 mg of poly(lactic-co-glycolic acid) (PLGA) isadded to pressure vessel (PV2). 1,1,1,2,3,3-hexafluoropropane is addedto the pressure vessel (PV2) through a valve and inlet. Pressure vessel(PV2) is kept at room temperature with no applied heat with the pressureinside the isolated vessel (PV2) approximately 4500 psi. Nozzle (P) isheated to 15° C.A 1-cm×2-cm L605 coupon is added to vessel (V) andconnected to a high-voltage power lead. Both nozzles (D) and (P) aregrounded. To begin, the coupon is charged to +7.5 kV after which port(3) connecting (PV) containing rapamycin to nozzle (D) charged at −7.5kV is opened allowing ejection of rapamycin into vessel (V) maintainedat ambient pressure. Alternatively, the substrate may be a stent oranother biomedical device as described herein, for example. Afterclosing port (3) and approximately 60-seconds, the metering valveconnecting (PV2) with nozzle (P) inside vessel (V) is opened allowingfor expansion of liquefied gas to a gas phase and introduction ofprecipitated polymer particles into vessel (V) while maintaining vessel(V) at ambient pressure. After approximately 15 seconds at a feed rateof approximately 3 cm³/min., the metering valve s closed while thecoupon remained charged. The sequential addition of drug followed bypolymer as described above is optionally repeated to increase the numberof drug-polymer layers after which the applied potential is removed fromthe coupon and the coupon was removed from the vessel. The coupon isthen examined using an optical microscope to to determine whether aconsistent coating is visible on all surfaces of the coupon except wherethe coupon was masked by the electrical lead.

Example 20 Dual Coating of a Metal Coupon with Crystalline Rapamycin andpoly(lactic-co-glycolic acid) (PLGA) Followed by SupercriticalHexafluoropropane Sintering

After inspection of the coupon created in example 19, the coated coupon(or other coated substrate, e.g. coated stent) is carefully placed in asintering vessel that is at a temperature of 75° C.1,1,1,2,3,3-hexafluoropropane in a separate vessel at 75 psi is slowlyadded to the sintering chamber to achieve a pressure of 23 to 27 psi.This hexafluoropropane sintering process is done to enhance the physicalproperties of the film on the coupon. The coupon remains in the vesselunder these conditions for approximately 10 min after which thesupercritical hexafluoropropane is slowly vented from the pressurevessel and then the coupon was removed and reexamined under an opticalmicroscope. The coating is observed in conformal, consistent, andsemi-transparent properties as opposed to the coating observed andreported in example 19 without dense hexafluoropropane treatment. Thecoated coupon is then submitted for x-ray diffraction (XRD) analysis,for example, as described herein to confirm the presence of crystallinerapamycin in the polymer.

Example 21 Coating of a Metal Cardiovascular Stent with CrystallineRapamycin and Poly(Lactic-Co-Glycolic Acid) (PLGA)

The apparatus described in examples 17, 18 and 20 is used in theforegoing example. The metal stent used is made from cobalt chromiumalloy of a nominal size of 18 mm in length with struts of 63 microns inthickness measuring from an abluminal surface to a luminal surface, ormeasuring from a side wall to a side wall. The stent is coated in analternating fashion whereby the first coating layer of drug is followedby a layer of polymer. These two steps, called a drug/polymer cycle, arerepeated twice so there are six layers in an orientation ofdrug-polymer-drug-polymer-drug-polymer. After completion of each polymercoating step and prior the application of the next drug coating step,the stent is first removed from the vessel (V) and placed in a smallpressure vessel where it is exposed to supercritical hexafluoropropaneas described above in example 20.

Example 22 Layered Coating of a Cardiovascular Stent with anAnti-Restenosis Therapeutic and Polymer in Layers to Control DrugElution Characteristics

A cardiovascular stent is coated using the methods described in examples10 and 11 above. The stent is coated in such as way that the drug andpolymer are in alternating layers. The first application to the barestent is a thin layer of a non-resorbing polymer, approximately2-microns thick. The second layer is a therapeutic agent withanti-restenosis indication. Approximately 35 micrograms are added inthis second layer. A third layer of polymer is added at approximately2-microns thick, followed by a fourth drug layer which is composed ofabout 25 micrograms of the anti-restenosis agent. A fifth polymer layer,approximately 1-micron thick is added to stent, followed by the sixthlayer that includes the therapeutic agent of approximately15-micrograms. Finally, a last polymer layer is added to a thickness ofabout 2-microns. After the coating procedure, the stent is annealedusing carbon dioxide as described in example 16 above. In this example adrug eluting stent (DES) is described with low initial drug “burst”properties by virtue of a “sequestered drug layering” process, notpossible in conventional solvent-based coating processes. Additionally,by virtue of a higher concentration of drug at the stent ‘inter-layer’the elution profile is expected to reach as sustained therapeuticrelease over a longer period of time.

Example 23 Layered Coating of a Cardiovascular Stent with anAnti-Restenosis Therapeutic and an Anti-Thrombotic Therapeutic in aPolymer

A cardiovascular stent is coated as described in example 11 above. Inthis example, after a first polymer layer of approximately 2-micronsthick, a drug with anti-thrombotic indication is added in a layer ofless than 2-microns in thickness. A third layer consisting of thenon-resorbing polymer is added to a thickness of about 4-microns. Nextanother drug layer is added, a different therapeutic, with ananti-restenosis indication. This layer contains approximately 100micrograms of the anti-restenosis agent. Finally, a polymer layerapproximately 2-microns in thickness is added to the stent. Aftercoating the stent is treated as described in example 20 to sinter thecoating using hexafluoropropane.

Example 24 Coating of Stent with Rapamycin and Poly(Lactic-Co-GlycolicAcid) (PLGA)

Micronized Rapamycin is purchased from LC Laboratories. 50:50 PLGA(Mw=˜90) are purchased from Aldrich Chemicals. Eurocor CoCr (7 cell)stents are used. The stents are coated by dry electrostatic capturefollowed by supercritical fluid sintering, using 3 stents/coating runand 3 runs/data set. Analysis of the coated stents is performed bymultiple techniques on both stents and coupons with relevant controlexperiments described herein.

In this example, PLGA is dissolved in 1,1,1,2,3,3-Hexafluoropropane withthe following conditions: a) room temperature, with no applied heat; b)4500 psi; and c) at 2 mg/ml concentration. The spray line is set at 4500psi, 150° C. and nozzle temperature at 150° C. The solvent(Hexafluoropropane) is rapidly vaporized when coming out of the nozzle(at 150° C.). A negative voltage is set on the polymer spray nozzle toachieve a current of greater than or equal to 0.02 mAmps. The stent isloaded and polymer is sprayed for 15 seconds to create a first polymercoating.

The stent is then transferred to a sintering chamber that is at 75° C.The solvent, in this example 1, 1,2,3,3-hexafluoropropane, slowly entersthe sintering chamber to create a pressure at 23 to 27 psi. Stents aresintered at this pressure for 10 minutes.

11.5 mg Rapamycin is loaded into the Drug injection port. The injectionpressure is set at 280 psi with +7.5 kV for the stent holder and −7.5 kVfor the drug injection nozzle. After the voltage is set for 60 s, thedrug is injected into the chamber to create a first drug coating.

A second polymer coating is applied with two 15 second sprays ofdissolved polymer with the above first polymer coating conditions. Thesecond coating is also subsequently sintered in the same manner.

A second drug coating is applied with the same parameters as the firstdrug coating. Lastly, the outer polymer layer is applied with three 15second sprays of dissolved polymer with the above polymer coatingconditions and subsequently sintered.

Example 25 Histology of In Vivo Stented Porcine Models and Preparationfor Pharmacokinetics Studies

Coronary stenting was applied to porcine animal models as describedpreviously. An angiography was perform on each animal prior toeuthanasia. After prenecropsy angiography, each animal was euthanizedvia an overdose of euthanasia solution or potassium chloride solution,IV in accordance to the Test Facility's Standard Operating Procedure andwas performed in accordance with accepted American Veterinary MedicalAssociation's “AVMA Guidelines on Euthanasia” (June 2007; accessed athttp://www.avma.org/issues/animal-welfare/euthansia.pdf). A limitednecropsy consisting of examination of the heart was performed on allanimals. Observations of macroscopic findings were recorded. Anyevidence of macroscopic findings, were processed for histologicalexamination. Regardless, all hearts were collected for histologicprocessing and assessment.

The hearts were perfusion fixed at ˜100 mmHg with Lactated Ringer'sSolution until cleared of blood followed by 10% neutral bufferedformalin (NBF). The fixed hearts were placed in a NBF filled containerand labeled as appropriate.

Whole heart radiographs were taken to document stent location andmorphology in situ. In addition, each explanted stent was radiographedin two views (perpendicular or orthogonal incidences) along itslongitudinal plane to assist in the assessment of expansion morphology,damage and/or areas of stent discontinuity (eg, strut fractures).

Fixed stented vessels were carefully dissected from the myocardium,leaving sufficient vessel both proximal and distal to the stentedportion. Unless otherwise stated or required, all tissues/sections wereprocessed according to the CBSET standard operating procedures. Inparticular, transverse sections of unstented vessel were obtained withinapproximately 1-3 mm of the proximal and distal ends of the stent (i.e.,unstented vessel) and from the proximal, middle and distal regions ofthe stented vessel. All vessel sections were stained with hematoxylinand eosin and a tissue elastin stain (e.g., Verhoeff's).

The remaining myocardium was then transversely sectioned (i.e.,“bread-loafed”) from apex to base (˜1 cm apart) to further assess forevidence of adverse reactions (e.g., infarction). If gross findings werepresent they were collected and processed for light microscopy.Remaining myocardial tissue were stored until finalization of the studyat which time, it was disposed of according to Test Facility standardoperating procedures, shipped to Sponsor, or archived at Sponsor'srequest and expense.

Quantitative morphometric analysis was performed on the histologicalsections from each stented artery. For each histological section, theparameters listed in Table 4 were directly measured using standard lightmicroscopy and computer-assisted image measurement systems.

TABLE 4 Morphometry Parameters Parameter Abbreviation Calculation UnitLumen Area L_(a) directly measured mm² Internal Elastic IEL_(a) directlymeasured mm² Layer (IEL) Bounded Area Stent Area S_(a) directly measuredmm² External Elastic EEL_(a) directly measured mm² Layer (EEL) BoundedArea

From these direct measurements, all other histomorphological parameterswere calculated. Measured and calculated parameters, formulae, and unitsof measure are given in Table 5.

TABLE 5 Calculated Morphometry Parameters and Units of Measure ParameterAbbreviation Calculation Unit Area Measurements Neointimal Area N_(a)IEL_(a) − L_(a) mm² Medial Area M_(a) EEL_(a) − IEL_(a) mm² Artery AreaA_(a) L_(a) + N_(a) + M_(a) mm² Length Measurements Lumen Diameter L_(d)2 × √(L_(a)/π) mm IEL Diameter IEL_(d) 2 × √(L_(a) + N_(a))/π mm StentDiameter S_(d) 2 × √(S_(a)/π) mm Arterial Diameter A_(d) 2 × √(A_(a)/π)mm Ratios Lumen/Artery L:A L_(a)/A_(a) NA* Areas Neointima/Media N:MN_(a)/M_(a) NA Areas EEL/IEL Areas EEL_(a):IEL_(a) A_(a)/(L_(a) + N_(a))NA IEL/Stent Areas IEL_(a):S_(a) IEL_(a)/S_(a) NA Restenosis Parameters% Area % AO N_(a) /(N_(a) + L_(a)) × 100% % Occlusions) Neointima N_(μm)N_(mm) × 1000(μm/mm) μm Thickness Neointima N_(mm) (IEL_(d) − L_(d))/2mm Thickness

Histopathology—Stented & Adjacent Non-Stented Vessels

Histopathological scoring via light microscopy was also used to gradevarious parameters that reflect the degree and extent of the hostresponse/repair process to treatment. These parameters included, butwere not limited to, injury, inflammation, endothelialization, andfibrin deposition. When a microscopic endpoint listed below is notpresent/observed, the score 0 was given.

The scoring of the arterial cross-sections was carried out as follows:

Injury score for stented arterial segments is dependent on that portionof the arterial wall which is disrupted by the stent and/or associatedtissue response. Injury was scored on a per-strut basis and the medianand average calculated per plane (i.e., proximal, middle, distal) andstent. The scoring polymer for injury at each strut is listed in Table6.

TABLE 6 Injury Score Polymer Score Value 0 IEL intact 1 Disruption ofIEL 2 Disruption of tunica media 3 Disruption of tunica adventitia

Inflammation score depends on the degree of inflammation and extent ofinflammation on a per-strut basis as outlined in Table 7. Inflammationwas scored on a per strut basis and the average was calculated per planeand stent.

TABLE 7 Inflammation Score Polymer Score Value 0 Absent 1 Scatteredcellular infiltrates associated with strut 2 Notable cellularinfiltrates associated with strut 3 Cellular infiltrates circumscribingstrutNeointimal fibrin score depends on the degree of fibrin deposition inthe neointima as outlined in Table 8.

TABLE 8 Neointimal Fibrin Score Polymer Score Value 0 Absent 1Infrequent spotting of fibrin 2 Heavier deposition of fibrin 3 Heavydeposition of fibrin that spans between strutsEndothelialization score depends on the extent of the circumference ofthe artery lumen showing coverage with endothelial cells as outlined inTable 9.

TABLE 9 Endothelialization Score Polymer Score Value 0 Absent 1 <25% 225% to 75% 3 >75% 4 100%, confluentAdventitial fibrosis score depends on the severity of response andcircumference of artery affected as outlined in Table 10.

TABLE 10 Adventitial Fibrosis Score Polymer Score Observation 0 Absent 1Minimal presence of fibrous tissue 2 Notable fibrous tissue in 25%-50%of artery circumference 3 Notable fibrous tissue in ≧50% of arterycircumferenceNeointimal maturation depends on the cellularity and organization of theneointima as outlined in Table 11.

TABLE 11 Neointimal Maturation Score Polymer Score Observation 0 Absent1 Immature, predominantly fibrino-vascular tissue 2 Transitional,predominantly organizing smooth muscle 3 Mature, generalized organizedsmooth muscle

The histologic section of the artery was also examined for otherhistologic parameters including, but not limited to, hemorrhage,necrosis, medial fibrosis, type and relative amounts of inflammatorycell infiltrates (eg, neutrophils, histiocytes, lymphocytes,multinucleated giant cells), mineralization, strut malapposition,thrombosis and/or neointimal vascularity, or others as deemedappropriate by the pathologist. Unless otherwise stated in the pathologydata/report, additional findings were graded as follow: 0=Absent;1=Present, but minimal feature; 2=Notable feature; 3=Overwhelmingfeature.

Sections of the non-stented proximal and distal portions of the stentedarteries, were similarly assessed and scored for histologic parametersas above (excluding neointimal fibrin) but were assessed forhistomorphometry.

One histology study according to the description above was performedusing the groups and coated stents (test articles) as noted in Table 12which were coated according to the methods provided herein, and/ordevices having coatings as described herein (for example, at AS1, AS2,or another coating combination as described herein) as compared to acontrol bare metal stent (BMS, AS3) The animals were Yucatan pigs, whichwere given an anticoagulation regimen of Day 1: ASA 650 mg+Plavix 300mg, maintenance of: ASA 81 mg+Plavix75, and Procedural: ACT ˜250 sec.Oversizing was 10-20%.

TABLE 12 Number of Necropsy Group Test Article Test Devices Time Point 1AS1 N = 6 Day 28 N = 6 Day 90 2 AS2 N = 6 Day 28 N = 6 Day 90 3 AS3(Bare N = 6 Day 28 metal Stent) N = 6 Day 90

A second histology study also according to the description above wasperformed and compared with a CYPHER stent control. In these studies,AS21, AS23, and AS24 were tested along with the CYPHER stent. AS21,AS23, and AS24 were designed with coatings comprising Polymer B asdescribed above, with about half the polymer load of AS1. AS23 and AS24had about half the amount of rapamycin as AS1, while AS21 was designedwith a target rapamycin load that was about the same as AS1, asdescribed previously.

Results of histology studies performed according to the methodsdescribed above are presented in FIGS. 12-23. FIGS. 12 and 13 depictlow-magnification cross-sections of porcine coronary artery stentimplants (AS1, AS2 and Bare-metal stent control) at 28 days and 90 dayspost-implantation. FIGS. 14 and 15 show drug depots in low-magnificationcross-sections of porcine coronary artery stent implants. FIG. 16 showsmean (n=3) sirolimus levels in arterial tissue following AS1 and Cypherstent implantation. The results for AS1 presented in FIG. 16 were takenfrom a separate study as the results for the Cypher Stents presented inFIG. 16. Both studies were performed as described above, and data wascollected similarly, however, data from the two studies were combined inthis Figure to illustrate a comparison for results obtained for AS1stent to results obtained for Cypher stent in a separate, but similarstudy. FIG. 17 shows mean sirolimus levels in arterial tissue followingvarious stent implantations. FIG. 18 shows arterial tissueconcentrations (y-axis) versus time (x-axis) for AS1 and AS2 stentsimplantations in swine coronary arteries expressed as absolute tissuelevel (y-axis) versus time (x-axis). FIG. 19 depicts mean (n=3)sirolimus levels in remaining on stent following various stentimplantations in swine coronary arteries expressed as stent level(y-axis) versus time (x-axis). FIG. 20 depicts mean (n=3) sirolimuslevels remaining on stent following AS1 and Cypher stent implantationsin swine coronary arteries expressed as stent level (y-axis) versus time(x-axis). The results for AS1 presented in FIG. 20 were taken from aseparate study as the results for the Cypher Stents presented in FIG.20. Both studies were performed as described above, and data wascollected similarly, however, data from the two studies were combined inthis Figure to show a comparison of results obtained for AS1 stent andresults obtained for the Cypher stent in a separate, but similar study.FIG. 21 is Fractional Sirolimus Release (y-axis) versus time (x-axis) inArterial Tissue for AS1 and AS2 Stents. FIG. 22 is sirolimus bloodconcentration following single stent implant expressed in bloodconcentration (ng/ml) (y-axis) versus time (x-axis). Pigs were implantedwith coated stents as described above. Blood was drawn at predeterminedtimes and assayed to determine rapamycin concentration. The assays werebased on technology known to one of ordinary skill in the art. FIG. 23shows mean (single stent normalized) blood concentration immediatelypost implant expressed as blood concentrations (ng/ml) (y-axis) for aCypher stent, and stents having coatings as described herein (AS21, AS1,AS23, AS24 are devices comprising coatings as described herein).

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. While embodiments of the presentinvention have been shown and described herein, it will be obvious tothose skilled in the art that such embodiments are provided by way ofexample only. Numerous variations, changes, and substitutions will nowoccur to those skilled in the art without departing from the invention.It should be understood that various alternatives to the embodiments ofthe invention described herein may be employed in practicing theinvention. It is intended that the following claims define the scope ofthe invention and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

1. A device comprising a. a stent; and b. a plurality of layers thatform a laminate coating on said stent; wherein at least one of saidlayers comprises a bioabsorbable polymer and at least one of said layerscomprises one or more active agents; wherein at least a portion of theactive agent is in crystalline form.
 2. A device comprising a. a stent;b. a plurality of layers that form a laminate coating on said stent;wherein at least one of said layers comprises a bioabsorbable polymerand at least one of said layers comprises a pharmaceutical agentselected from rapamycin, a prodrug, a derivative, an analog, a hydrate,an ester, and a salt thereof, wherein at least a portion of thepharmaceutical agent is in crystalline form.
 3. The device of claim 2,wherein said device has at least one pharmaceutical agent layer definedby a three-dimensional physical space occupied by crystal particles ofsaid pharmaceutical agent and said three dimensional physical space isfree of polymer.
 4. The device of claim 3, wherein at least some of thecrystal particles in said three dimensional physical space defining saidat least one pharmaceutical agent layer are in contact with polymerparticles present in a polymer layer adjacent to said at least onepharmaceutical agent layer defined by said three-dimensional space freeof polymer.
 5. The device of claim 2, wherein said plurality of layerscomprises a first polymer layer comprising a first bioabsorbable polymerand a second polymer layer comprising a second bioabsorbable polymer,wherein said at least one layer comprising said pharmaceutical agent isbetween said first polymer layer and said second polymer layer.
 6. Thedevice of claim 5, wherein said first and second bioabsorbable polymersare the same polymer.
 7. The device of claim 5, wherein said first andsecond bioabsorbable polymers are different.
 8. The device of claim 5,wherein said second polymer layer has at least one contact point with atleast one particle of said pharmaceutical agent in said pharmaceuticalagent layer and said second polymer layer has at least one contact pointwith said first polymer layer.
 9. The device of claim 8 wherein saidstent has a stent longitudinal axis; and said second polymer layer has asecond polymer layer portion along said stent longitudinal wherein saidsecond layer portion is free of contact with particles of saidpharmaceutical agent.
 10. The device of claim 9, wherein said device hasat least one pharmaceutical agent layer defined by a three-dimensionalphysical space occupied by crystal particles of said pharmaceuticalagent and said three dimensional physical space is free of polymer. 11.The device of claim 9, wherein said stent comprises at least one struthaving a strut length along said stent longitudinal axis, wherein saidsecond layer portion extends substantially along said strut length. 12.The device of claim 9, wherein said stent has a stent length along saidstent longitudinal axis and said second layer portion extendssubstantially along said stent length.
 13. The device of claim 9,wherein said stent comprises at least five struts, each strut having astrut length along said stent longitudinal axis, wherein said secondlayer portion extends substantially along substantially the strut lengthof at least two struts.
 14. The device of claim 9, wherein said stentcomprises at least five struts, each strut having a strut length alongsaid stent longitudinal axis, wherein said second layer portion extendssubstantially along substantially the strut length of at least threestruts.
 15. The device of claim 9, wherein said stent comprises at leastfive struts, each strut having a strut length along said stentlongitudinal axis, wherein said second layer portion extendssubstantially along substantially the strut length of least four struts.16. The device of claim 9, wherein said stent comprises at least fivestruts, each strut having a strut length along said stent longitudinalaxis, wherein said second layer portion extends substantially alongsubstantially the strut length of all said at least five struts.
 17. Thedevice of claim 9, wherein said stent has a stent length along saidstent longitudinal axis and said second layer portion extendssubstantially along said stent length.
 18. The device of claim 9,wherein said stent has a stent length along said stent longitudinal axisand said second layer portion extends along at least 50% of said stentlength.
 19. The device of claim 9, wherein said stent has a stent lengthalong said stent longitudinal axis and said second layer portion extendsalong at least 75% of said stent length.
 20. A device comprising: a. astent; b. a plurality of layers that form a laminate coating on saidstent, wherein a first layer comprises a first bioabsorbable polymer, asecond layer comprises a pharmaceutical agent, a third layer comprises asecond bioabsorbable polymer, a fourth layer comprises thepharmaceutical agent, and a fifth layer comprises a third bioabsorbablepolymer, wherein the pharmaceutical agent is selected from rapamycin, aprodrug, a derivative, an analog, a hydrate, an ester, and a saltthereof, and wherein at least a portion of the pharmaceutical agent isin crystalline form.